Stabilization and isolation of extracellular nucleic acids

ABSTRACT

The present invention provides methods, compositions and devices for stabilizing the extracellular nucleic acid population in a cell-containing biological sample using a poly(oxyethylene) polymer or mono-ethylene glycol as stabilizing agent.

FIELD OF THE INVENTION

The technology disclosed herein relates to methods and compositionssuitable for stabilizing the extracellular nucleic acid population in acell-containing sample, in particular a blood sample, and to a methodfor isolating extracellular nucleic acids from respectively stabilizedbiological samples.

BACKGROUND

Extracellular nucleic acids have been identified in blood, plasma, serumand other body fluids. Extracellular nucleic acids that are found inrespective samples are to a certain extent degradation resistant due tothe fact that they are protected from nucleases (e.g. because they aresecreted in form of a proteolipid complex, are associated with proteinsor are contained in vesicles). The presence of elevated levels ofextracellular nucleic acids such as DNA and/or RNA in many medicalconditions, malignancies, and infectious processes is of interest interalia for screening, diagnosis, prognosis, surveillance for diseaseprogression, for identifying potential therapeutic targets, and formonitoring treatment response.

Additionally, elevated fetal DNA/RNA in maternal blood is being used todetermine e.g. gender identity, assess chromosomal abnormalities, andmonitor pregnancy-associated complications. Thus, extracellular nucleicacids are in particular useful in non-invasive diagnosis and prognosisand can be used e.g. as diagnostic markers in many fields ofapplication, such as non-invasive prenatal genetic testing, oncology,transplantation medicine or many other diseases and, hence, are ofdiagnostic relevance (e.g. fetal- or tumor-derived nucleic acids).However, extracellular nucleic acids are also found in healthy humanbeings. Common applications and analysis methods of extracellularnucleic acids are e.g. described in WO97/035589, WO97/34015, Swarup etal, FEBS Letters 581 (2007) 795-799, Fleischhacker Ann. N.Y. Acad. Sci.1075: 40-49 (2006), Fleischhacker and Schmidt, Biochmica et BiophysicaActa 1775 (2007) 191-232, Hromadnikova et al (2006) DNA and Cellbiology, Volume 25, Number 11 pp 635-640; Fan et al (2010) ClinicalChemistry 56:8.

Extracellular nucleic acids are usually only comprised in a lowconcentration in the samples. E.g. free circulating nucleic acids arepresent in plasma in a concentration of 1-100 ng/ml plasma. Furthermore,extracellular nucleic acids often circulate as fragments of a size of500 nt, 300 nt (when indicating the size and hence the chain length theterm “nt” also includes “bp” in case of DNA) or even less (circulatingnucleosomes). For ccf DNA in plasma, the average length is often onlyapprox. 140-170 bp. Additionally, the actual target extracellularnucleic acid that is supposed to be identified for diagnostic purposesusually also represents only a small fraction among the totalextracellular nucleic acids. With respect to ccfDNA, usually only a fewthousand amplifiable copies are present per ml of blood depending e.g.on the pregnancy state or tumor grade. Specifically tumor specific DNAfragments are very rare and often are comprised in a concentration thatis 1000-fold less than the “normal” extracellular nucleic acidbackground. This low concentration poses challenges with respect to thestabilization of the sample and the subsequent isolation of theextracellular nucleic acids from the stabilized samples.

A major problem regarding the analysis of circulating, cell-free nucleicacids (cf NA) such as from tumors or of foetal origin is—besides thedegradation that occurs in serum and probably also plasma—the possibledilution of extracellular DNA (and RNA) by genetic material from damagedor decaying cells after sample collection. After the sample wascollected, cellular nucleic acid are released from the cells containedin the sample due to cell breakage during ex vivo incubation, typicallywithin a relatively short period of time after sample collection. Oncecell lysis begins, the lysed cells release large amounts of additionalnucleic acids which become mixed with the extracellular nucleic acidsand it becomes increasingly difficult to recover the extracellularnucleic acids for testing. With respect to blood samples, in particularthe lysis of white blood cells is a problem as they release largeamounts of genomic DNA in addition to RNA. Red blood cells do notcontain genomic DNA. Therefore, stabilization of circulating nucleicacids in whole blood must include mechanism to stabilize blood cells inorder to prevent during stabilization a contamination of theextracellular nucleic acid population by cellular genomic DNA and alsoRNA. In particular the dilution of the extracellular nucleic acids, inparticular rare target extracellular nucleic acids, is an issue and mustbe prevented. These problems are discussed in the prior art (see e.g.Chiu et al (2001), Clinical Chemistry 47:9 1607-1613; Fan et al (2010)and US2010/0184069). Further, the amount and recoverability of availableextracellular nucleic acids can decrease substantially over a period oftime due to degradation. Besides mammalian extracellular nucleic acidsthat derive e.g. from tumor cells or the fetus, cell-containing samplesmay also comprise other nucleic acids of interest that are not comprisedin cells. An important, non-limiting example is pathogen nucleic acidssuch as viral nucleic acids. Preservation of the integrity of viralnucleic acids in cell-containing samples such as in particular in bloodspecimens during shipping and handling is also crucial for thesubsequent analysis and viral load monitoring.

The release from intracellular nucleic acids after sample collectionparticularly is an issue, if the sample comprises a high amount of cellsas is the case e.g. with whole blood samples. Thus, in order to avoidrespectively reduce the above described problems it is common toseparate an essentially cell-free fraction of the sample from the cellscontained in the sample basically immediately after the sample isobtained. E.g. it is recommended to obtain blood plasma from whole bloodbasically directly after the blood is drawn and/or to cool the wholeblood and/or the obtained plasma or serum in order to preserve theintegrity of the extracellular nucleic acids and to avoid contaminationsof the extracellular nucleic acid population with intracellular nucleicacids that are released from the contained cells. However, obtaining anessentially cell-free fraction of a sample can be problematic and theseparation is frequently a tedious and time consuming multi-step processas it is important to use carefully controlled conditions to preventcell breakage during centrifugation which could contaminate theextracellular nucleic acids with cellular nucleic acids released duringbreakage. The need to directly separate e.g. the plasma from the bloodis also a major disadvantage with respect to the handling of the samplesas appropriate equipment is not necessarily available at the site wherethe sample is collected. Furthermore, it is often difficult to removeall cells. Thus, many processed samples that are often and commonlyclassified as “cell-free” such as plasma or serum in fact still containresidual amounts of cells that were not removed during the separationprocess. These cells may also become damaged or may die during handlingof the sample, thereby releasing intracellular nucleic acids, inparticular genomic DNA, as is described above. These remaining cellsalso pose a risk that they become damaged during the handling so thattheir nucleic acid content, particularly genomic (nuclear) DNA andcytoplasmic RNA, would merge with and thereby contaminate respectivelydilute the extracellular, circulating nucleic acid fraction. To removethese remaining contaminating cells and to avoid/reduce theaforementioned problems, it was known to perform a second centrifugationstep at higher speed. However, again, such powerful centrifuges areoften not available at the facilities wherein the blood is obtained.Furthermore, even if plasma is obtained directly after the blood isdrawn, it is recommended to freeze it at −80° C. in order to preservethe nucleic acids contained therein if the nucleic acids can not bedirectly isolated. This too imposes practical constraints upon theprocessing of the samples as e.g. the plasma samples must be shippedfrozen. This increases the costs and furthermore, poses a risk that thesample gets compromised in case the cold chain is interrupted.

With respect to the stabilization of blood, the following technologiesare known in the art: Blood samples are usually collected in bloodcollection tubes containing spray-dried or liquid EDTA (e.g. BDVacutainer K₂EDTA). EDTA chelates magnesium, calcium and other bivalentmetal ions, thereby inhibiting enzymatic reactions, such as e.g. bloodclotting or DNA degradation due to DNases. However, EDTA does notefficiently prevent the dilution respectively contamination of theextracellular nucleic acid population by released intracellular nucleicacids during storage. Thus, the extracellular nucleic acid populationthat is found in the cell-free portion of EDTA stabilised sampleschanges during the storage and becomes contaminated with large amountsof intracellular nucleic acids, in particular genomic DNA. Accordingly,EDTA is not capable of sufficiently stabilizing the extracellularnucleic acid population in particular because it can not avoid thecontamination of the extracellular nucleic acid population with e.g.genomic DNA fragments which are generated after blood draw by celldegradation and cell instability during sample transportation andstorage.

Blood collection tubes are known that contain reagents for an immediatestabilization of the RNA gene expression profile and thus thetranscriptome at the point of sample collection (see for example U.S.Pat. No. 6,617,170, U.S. Pat. No. 7,270,953, Kruhoffer et al, 2007).However, these methods are based on the immediate lysis of the cellscontained in the sample. Therefore, these methods and other methods thatinduce cell lysis are unsuitable for stabilizing the extracellularnucleic acid population in a cell-containing sample, because they inducethe release of intracellular nucleic acids which become thereby mixedwith the extracellular nucleic acid population.

Furthermore, methods are known in the prior art for stabilizingcell-containing samples, such as blood or tissue samples, whichstabilize e.g. the cells, the transcriptome, genome and proteome. Such amethod is e.g. disclosed in WO 2008/145710. Said method is based on theuse of specific stabilizing compounds, such as for exampleN,N-Dimetyhlacetamide. However, N,N-dimetyhlacetamide is a toxic agent.Therefore, there is a need to provide alternative stabilization methodswhich avoid the use of toxic agents.

Methods are known in the prior art that specifically aim at stabilizingextracellular nucleic acids contained in whole blood. One method employsthe use of formaldehyde to stabilize the cell membranes, therebyreducing the cell lysis and furthermore, formaldehyde inhibitsnucleases. Respective methods are e.g. described in U.S. Pat. No.7,332,277 and U.S. Pat. No. 7,442,506. To address the need ofsimultaneous cell stabilization and nucleic acid stabilization,stabilization systems were developed that are based on the use offormaldehyde releasers. Respective stabilization agents are commerciallyavailable from Streck Inc. under the name of cell-free RNA BCT (bloodcollection tube). The 10 ml blood collection tube is intended for thepreservation and stabilization of cell-free RNA in plasma for up to 3days at room temperature. The preservative stabilizes cell-free RNA inplasma and prevents the release of non-target background RNA from bloodcells during sample processing and storage. US 2011/0111410 describesthe use of formaldehyde releasing components to achieve cell and RNAstabilization in the same blood sample. Therefore, this documentdescribes a technique wherein the stabilization agent stabilises theblood cells in the drawn blood thereby preventing contamination ofcellular RNA with cell-free RNA or globin RNA, inhibits the RNAsynthesis for at least 2 hours and cellular RNA that is within the bloodcells is preserved to keep the protein expression pattern of the bloodcells substantially unchanged to the time of the blood draw. The whiteblood cells can be isolated from the respectively stabilised sample andcellular RNA is than extracted from the white blood cells. However, theuse of formaldehyde or formaldehyde-releasing substances has drawbacks,as they compromise the efficacy of extracellular nucleic acid isolationby induction of crosslinks between nucleic acid molecules or betweenproteins and nucleic acids. Methods to stabilize blood samples are alsodescribed e.g. in US 2010/0184069 and US 2010/0209930.

PCT/EP2012/070211 and PCT/EP2012/068850 describe different methods forstabilizing the extracellular nucleic acid population in acell-containing biological sample such as a whole blood sample. Thestabilization compositions described in these applications are effectivein stabilizing the extracellular nucleic acid population, in particularby preventing the release of intracellular nucleic acids into theextracellular nucleic acid population.

There is a continuous need to develop and improve methods that result ina stabilization of the extracellular nucleic acid population comprisedin a cell-containing biological sample, including samples suspected ofcontaining cells, in particular whole blood, plasma or serum, therebymaking the handling, respectively processing of such samples easier. Byproviding efficient and reliable sample stabilization technologies whichpreferably do not impair the subsequent nucleic acid isolation, theisolation and testing of extracellular nucleic acids contained in suchsamples becomes more reliable and consequently, the diagnostic andprognostic application/use of extracellular nucleic acids is improved bysuch stabilization technologies. In particular, there is a continuousneed for a solution for preserving the extracellular nucleic acidpopulation in whole blood samples, e.g. for prenatal testing and/or forscreening for diseases such as e.g. neoplastic, in particularpremalignant or malignant diseases.

It is the object of the present invention to provide methods andcomposition for stabilizing the extracellular population comprised in acell-containing sample. In particular, it is the object to overcome atleast one of the drawbacks of the prior art sample stabilizationmethods. Furthermore, it is in particular an object of the presentinvention to provide a method suitable for stabilizing a cell-containingbiological sample, in particular a whole blood sample, at roomtemperature. Furthermore, it is an object of the present invention toprovide a sample collection container, in particular a blood collectiontube, that is capable of effectively stabilizing a cell-containingbiological sample and in particular is capable of stabilizing theextracellular nucleic acid population comprised in the sample.Furthermore, it is one object of the present invention to provide astabilization technology which subsequently allows isolation of theextracellular nucleic acids from the stabilized sample with good yield.

SUMMARY OF THE INVENTION

The present invention is based on the surprising finding thatpoly(oxyethylene) polymers such as polyethylene glycol are effective instabilizing cell-containing biological samples comprising extracellularnucleic acids, in particular whole blood samples. It was found thatpoly(oxyethylene) polymers such as polyethylene glycol of differentmolecular weights and in various concentrations are capable ofstabilizing the extracellular nucleic acid population of thecell-containing sample and in particular are capable to reduce the riskthat the extracellular nucleic acid population becomes contaminated withgenomic DNA, in particular fragmented genomic DNA, after the sample wascollected. Using a poly(oxyetlylene) polymer such as polyethylene glycolas stabilizing agent reduces the risk that the extracellular nucleicacid population becomes diluted by intracellular nucleic acids such asin particular genomic DNA, what significantly contributes to thepreservation of the profile of the extracellular nucleic acid populationat the time of sample collection. The stabilization effect was strongerthan that seen with many other stabilization agents. Furthermore, it wasfound that poly(oxyethylene) polymers such as preferably polyethyleneglycol can be advantageously used in combination with other stabilizingagents such as caspase inhibitors and amides in order to further improvethe stabilization effect on the cell-containing sample. Advantageously,poly(oxyethylene) polymers such as the preferred polyethylene glycol arenot classified as toxic, harmful or irritant agent. In advantageousembodiments, two or more poly(oxyethylene) glycols are used that differin their molecular weight. Preferably, a high molecular weightpoly(oxyethylene) polymer having a molecular weight of at least 1500 isused in combination with a low molecular weight poly(oxyethylene)polymer having a molecular weight of 1000 or less for stabilization. Itwas found that a balanced combination of poly(oxyethylene) polymers thatdiffer in their molecular weight, such as using a high and low molecularweight poly(oxyethylene) polymer, provides efficiently stabilizedsamples, from which the extracellular nucleic acids can be subsequentlyisolated with good yield using various standard nucleic acid isolationmethods. Furthermore, the use of mono-ethylene glycol (1,2-ethanediol)as stabilizing agent for cell-containing samples is described herein.Mono-ethylene gylcol may also be used in combination with thepoly(oxyethylene) polymer for stabilization.

According to a first aspect, a method suitable for stabilizing anextracellular nucleic acid population comprised in a cell-containingbiological sample is provided, comprising contacting the cell-containingsample with at least one poly(oxyethylene) polymer as stabilizing agentor with mono-ethylene glycol as stabilizing agent.

According to a second aspect, a method for isolating extracellularnucleic acids from a cell-containing biological sample is provided,wherein said method comprises

-   a) stabilizing the cell-containing biological sample according to    the method defined in the first aspect of the present invention; and-   b) isolating extracellular nucleic acids from the stabilized sample.

According to a third aspect, a composition suitable for stabilizing acell-containing biological sample is provided comprising

-   i) a poly(oxyethylene) polymer as stabilizing agent or-   ii) mono-ethylene glycol as stabilizing agent    and one or more, preferably two or more further additives selected    from the group consisting of    -   one or more primary, secondary or tertiary amides;    -   a caspase inhibitor;    -   an anticoagulant and/or a chelating agent.

Preferably, the composition according to the third aspect comprises apoly(oxyethylene) polymer, which preferably is a high molecular weightpoly(oxyethylene) polymer having a molecular weight of at least 1500, asstabilizing agent and furthermore comprises one or more, preferably twoor more further additives selected from the group consisting of

-   -   at least one further poly(oxyethylene) polymer having a        molecular weight that is at least 100, preferably at least 200,        at least 300 or at least 400 below the molecular weight of the        first poly(oxyethylene) polymer, which preferably is a high        molecular weight poly(oxyethylene) polymer, wherein said further        poly(oxyethylene) polymer preferably is a low molecular weight        poly(oxyethylene) polymer having a molecular weight of 1000 or        less;    -   one or more primary, secondary or tertiary amides;    -   a caspase inhibitor;    -   an anticoagulant and/or a chelating agent.

A respective stabilizing composition is particularly effective instabilizing a cell-containing biological sample, such as blood, plasmaand/or serum, by stabilizing cells and the extracellular nucleic acidpopulation comprised in said sample. The extracellular nucleic acidpopulation contained in the cell-containing biological sample issubstantially preserved over the stabilization period in the state ithad shown at the time the biological sample was contacted with saidstabilizing composition. The release of genomic DNA and otherintracellular nulceic acids is significantly reduced as is shown by theexamples. Extracellular nucleic acids isolated from respectivelystabilized samples comprise significantly less contamination withintracellular nucleic acids, in particular fragmented genomic DNA,compared to extracellular nucleic acids that are isolated fromunstabilized samples. A respective stabilizing composition allows thestorage and/or handling, e.g. shipping, of the stabilized sample, e.g.blood, at room temperature for days without substantially compromisingthe quality of the sample, respectively the extracellular nucleic acidpopulation contained therein.

According to a fourth aspect, the present invention is related to theuse of the composition according to third aspect for stabilizing theextracellular nucleic acid population in a cell-containing biologicalsample, preferably a blood sample.

According to a fifth aspect, a collection device for collecting acell-containing biological sample is provided, wherein the collectiondevice comprises

-   -   i) a poly(oxyethylene) polymer as stabilizing agent or    -   ii) mono-ethylene glycol as stabilizing agent        and one or more further additives selected from the group        consisting of    -   one or more primary, secondary or tertiary amides;    -   a caspase inhibitor;    -   an anticoagulant and/or a chelating agent.

Preferably, the collection device according to the fifth aspectcomprises a poly(oxyethylene) polymer, which preferably is a highmolecular weight poly(oxyethylene) polymer having a molecular weight ofat least 1500, as stabilizing agent and furthermore comprises one ormore, preferably two or more further additives selected from the groupconsisting of

-   -   at least one further poly(oxyethylene) polymer having a        molecular weight that is at least 100, preferably at least 200,        at least 300 or at least 400 below the molecular weight of the        first poly(oxyethylene) polymer which preferably is a high        molecular weight poly(oxyethylene) polymer, wherein said further        poly(oxyethylene) polymer preferably is a low molecular weight        poly(oxyethylene) polymer having a molecular weight of 1000 or        less;    -   one or more primary, secondary or tertiary amides;    -   a caspase inhibitor;    -   an anticoagulant and/or a chelating agent.

The collection device for collecting a cell-containing biologicalsample, preferably a blood sample, may comprise a stabilizingcomposition according to the third aspect of the present invention.Providing a respective collection device, e.g. a sample collection tube,comprising the stabilizing composition has the advantage that thecell-containing biological sample is immediately stabilized as soon asthe sample is collected in the respective device.

According to a sixth aspect, a method is provided comprising the step ofcollecting, preferably drawing, a biological sample, preferably blood,from a patient directly into a chamber of a container according to thefifth aspect of the present invention.

According to a seventh aspect, a method of producing a compositionaccording to the third aspect of the present invention is provided,wherein the components of the composition are mixed, preferably aremixed in a solution. The term “solution” as used herein in particularrefers to a liquid composition, preferably an aqueous composition. Itmay be a homogenous mixture of only one phase but it is also within thescope of the present invention that a solution comprises solidcomponents such as e.g. precipitates.

Other objects, features, advantages and aspects of the presentapplication will become apparent to those skilled in the art from thefollowing description and appended claims. It should be understood,however, that the following description, appended claims, and specificexamples, while indicating preferred embodiments of the application, aregiven by way of illustration only.

DETAILED DESCRIPTION OF THIS INVENTION

The present invention is directed to methods, compositions and devicesand thus to technologies for stabilizing the extracellular nucleic acidpopulation comprised in a cell-containing biological sample that arebased on the use of a poly(oxyethylene) polymer as stabilizing agent. Itis shown that poly(oxyethylene) polymers such as polyethylene glycol ofdifferent molecular weights and in various concentrations are effectiveas stabilizing agents. Furthermore, advantageous combinations with otherstabilizing agents are described. The stabilization that is achievedwith the methods and compositions of the present invention allows thestorage and/or handling of the stabilized sample for a prolonged periodof time at room temperature.

The stabilization technologies described herein reduce the risk that theextracellular nucleic acid population comprised in the cell-containingsample becomes contaminated and thus becomes diluted with intracellularnucleic acids, in particular fragmented genomic DNA, originating fromdamaged and/or dying cells contained in the sample. As the compositionof the extracellular nucleic acid population is stabilized and thus issubstantially preserved at the time the sample is obtained, the timebetween sample collection and nucleic acid isolation can vary within thesuitable stabilization period without significant negative effect on thecomposition of the extracellular nucleic acid population. This is animportant advantage as it reduces variabilities in the extracellularnucleic acid population attributable to different handling procedures.Extracellular nucleic acids isolated from respectively stabilizedsamples comprise significantly less contamination with intracellularnucleic acids, in particular fragmented genomic DNA, compared toextracellular nucleic acids that are isolated from unstabilized samples.The described stabilization technologies improve the standardization ofdiagnostic or prognostic extracellular nucleic acid analyses becausevariations in the handling/storage of the samples have less influence onthe quality, respectively the composition and thus profile of theextracellular nucleic acid population comprised in the cell-containingbiological sample, thereby making diagnostic or prognostic applicationsthat are based on the extracellular nucleic acid fraction more reliableand more independent from the used storage/handling conditions. Theachieved substantial preservation is an important advantage because itenhances the accuracy of any subsequent tests that aims at analysingextracellular nucleic acids. Thereby, the diagnostic and prognosticapplicability of the respectively isolated extracellular nucleic acidsis improved. Specific embodiments described herein have the advantagethat the ratio of certain extracellular nucleic acid molecules comprisedin the population of extracellular nucleic acids can be kept moreconstant and thus more comparable to the ratio present at the time thebiological sample was collected. Thus, advantageously, the profile ofthe extracellular nucleic acid population can be preserved.

Hence, the analysis of respective cell containing biological samples,respectively the extracellular nucleic acids obtained from respectivelystabilized samples, becomes more comparable.

Furthermore, the teachings of the present invention obviate thenecessity to directly separate cells contained in the biological samplefrom the cell-free portion of the sample after sample collection inorder to avoid, respectively reduce, contaminations of the extracellularnucleic acids with intracellular nucleic acids, in particular fragmentedgenomic DNA, that is otherwise released from cells that die or decayduring storage/shipping. This advantage considerably simplifies thehandling of cell-containing biological samples, such as whole bloodsamples. However, the teachings of the invention are also advantageouswhen processing cell-depleted biological samples, or samples commonlyreferred to as being “cell-free” such as e.g. blood plasma or serum.Respective cell-depleted or “cell-free” biological samples may still(also depending on the used separation process) comprise residual cells,in particular white blood cells which comprise genomic DNA. Saidresidual cells pose a risk that the extracellular nucleic acidpopulation becomes increasingly contaminated with intracellular nucleicacids, in particular fragmented genomic DNA, if the (potentially)remaining cells are damaged or die during the shipping or storingprocess. This risk is considerably reduced when using the stabilizationmethods taught by the present invention. Thus, the present invention hasmany advantages when stabilizing biological samples which comprise largeamounts of cells such as e.g. blood samples, but also has importantadvantages when stabilizing biological samples which comprise smaller oronly a small amount of cells or which may only be suspected ofcontaining cells such as e.g. plasma, serum, urine, saliva, synovialfluids, amniotic fluid, lachrymal fluid, lymphatic fluid, liquor,cerebrospinal fluid and the like.

Furthermore, the use of mono-ethylene glycol is described forstabilizing the extracellular nucleic acid population in acell-containing sample, wherein optionally, mono-ethylene glycol is usedin combination with the poly(oxyethylene) polymer and/or one or more ofthe further stabilizing agents described herein.

A. Method of Stabilization

According to a first aspect, a method suitable for stabilizing anextracellular nucleic acid population comprised in a cell-containingbiological sample is provided which comprises a step of contacting thecell-containing biological sample with at least one poly(oxyethylene)polymer as stabilizing agent or with mono-ethylene glycol as stabilizingagent. The advantages of using a poly(oxyethylene) polymer asstabilizing agent were described above.

Preferably, the method according to the first aspect comprises a step ofcontacting the cell-containing biological sample with at least onepoly(oxyethylene) polymer as stabilizing agent.

The term poly(oxyethylene) polymer in particular refers to an anoligomer or polymer of ethylene oxide. It comprises at least twoethylene oxide units. Poly(oxyethylene) polymers are known in low andhigh molecular weights. Their molecular weight are usually multitutes of44, the molecular weight of its monomer, and can range up to 100000. Themolecular weight is indicated in Da. The poly(oxyethylene) polymer maybe linear or branched or may have other geometries. A linearpoly(oxyethylene) polymer is preferred. The poly(oxyethylene) polymermay be unsubstituted or substituted and preferably is polyethyleneglycol. As is demonstrated in the examples, polyethylene glycol has invarious molecular weights and in various concentrations a stabilizationeffect on cells and therefore can be used alone or in combination withother stabilization agents in order to stabilize the extracellularnucleic acid population in a cell-containing sample, in particular byreducing dilution of the extracellular nucleic acid population byintracellular nucleic acids such as in particular genomic DNA. However,also other poly(oxyethylene) polymers may be used that achieve astabilization effect as was shown for polyethylene glycol. As mentioned,also substituted poly(oxyethylene) polymers having a stabilizing effectmay be used such as alkyl poly(oxyethylene) polymers, e.g.alkylpolyethylene glycols, but also poly(oxyethylene) esters,poly(oxyethylene) amines, poly(oxyethylene) thiol compounds,poly(oxyethylene) glycerides and others. The preferred embodiment of thepoly(oxyethylene) polymer that is used as stabilizing agent ispolyethylene glycol. It preferably is unbranched and may beunsubstituted or substituted. Known substituted forms of polyethyleneglycol include alkylpolyethylene glycols that are e.g. substituted atone or both ends with an C1-C5 alkyl group. Preferably, unsubstitutedpolyethylene glycol of the formula HO—(CH₂CH₂O)_(n)—H is used. Alldisclosures described in this application for the poly(oxyethylene)polymer in general, specifically apply and particularly refer to thepreferred embodiment polyethylene glycol even if not explicitly stated.The poly(oxyethylene) polymer can be used in various molecular weights.Preferably, the term polyethylene glycol refers to oligo or polymers asis also evident from the molecular weights specified herein as suitableand preferred for the poly(oxyethylene) polymer which specifically alsoapply to the preferred embodiment polyethylene glycol.

A correlation was found between the stabilization effect of thepoly(oxyethylene) polymer and its molecular weight. Higher molecularweight poly(oxyethylene) polymers were found to be more effectivestabilizing agents than lower molecular weight poly(oxyethylene)polymers. To achieve an efficient stabilization with a lower molecularweight poly(oxyethylene) polymer, generally higher concentrations arerecommendable compared to a higher molecular weight poly(oxyethylene)polymer. For some applications such as blood samples it is preferredthough to keep the amount of additives used for stabilization low.Therefore, in embodiments, higher molecular weight poly(oxyethylene)polymers are used as stabilizing agents, as they allow to use lowerconcentrations of the poly(oxyethylene) polymer while achieving a strongstabilization effect on the extracellular nucleic acid population.

Thus, according to one embodiment, a high molecular weightpoly(oxyethylene) polymer having a molecular weight of at least 1500 isused as stabilizing agent and is contacted with the cell-containingsample. The high molecular weight poly(oxyethylene) polymer may have amolecular weight that lies in a range selected from 1500 to 50000, 1500to 40000, 2000 to 40000, 3000 to 40000, 2000 to 30000, 2500 to 30000,2500 to 25000, 3000 to 20000, 4000 to 20000, 3500 to 15000, 3000 to10000, 4000 to 10000, 4500 to 10000, 4500 to 9000, 4500 to 8000, 5000 to8000, 5000 to 7000 and 5500 to 7000. As is demonstrated by the examples,many different high molecular weight poly(oxyethylene) polymers can beused in conjunction with the invention. Suitable high molecular weightpoly(oxyethylene) polymers are also described in conjunction withdifferent aspects and embodiments of the invention. These molecularweights are particularly preferred for the use of a polyethylene glycol,in particular an unsubstituted polyethylene glycol. Unsubstitutedpolyethylene glycol was also used in the examples. The molecular weightof a poly(oxyethylene) polymer having a specific molecular weight mayvary within certain ranges conditional to manufacturing as is well-knownto the skilled person.

The high molecular weight poly(oxyethylene) polymer is used in aconcentration, wherein it exerts or supports the stabilization of theextracellular nucleic acid population that is contained in thecell-containing sample. Suitable concentrations for different sampletypes can be determined by the skilled person, for example by testingdifferent concentrations of a specific high molecular weightpoly(oxyethylene) polymer in the test assays described in the examples.As is demonstrated by the examples, the high molecular weightpoly(oxyethylene) polymer is effective in various concentrations. Theachieved stabilization effect and the preferred concentration alsodepends on whether one or more additional stabilizing agents are used.Preferred combinations are described herein. According to oneembodiment, the mixture that is obtained after contacting thecell-containing biological sample with the high molecular weightpoly(oxyethylene) polymer and optionally further additives comprises thehigh molecular weight poly(oxyethylene) polymer in a concentration rangethat is selected from 0.05% to 4% (w/v), 0.1% to 3% (w/v), 0.2% to 2.5%(w/v), 0.25% to 2% (w/v), 0.3% to 1.75% (w/v) and 0.35% to 1.5% (w/v).According to one embodiment, the high molecular weight poly(oxyethylene)polymer is used in lower concentration ranges such as 0.25% to 1.5%(w/v), 0.3% to 1.25% (w/v), 0.35% to 1% (w/v) and 0.4% to 0.75% (w/v).The above concentrations ranges are particularly suitable for thestabilization of blood. Using a high molecular weight poly(oxyethylene)polymer in a concentration of 1.5% (w/v) or less, 1.25% (w/v) or less,1% (w/v) or less and in particular in a concentration of 0.75% (w/v) orless is advantageous in certain embodiments. It was found in embodimentswherein a high molecular weight poly(oxyethylene) polymer was used incertain higher concentrations in the resulting mixture comprising thestabilizing agent and the cell-containing sample, such as a bloodsample, the subsequent isolation of the extracellular nucleic acids maybecome impaired when using certain standard nucleic acid isolationprocedures that involve e.g. the use of silica columns. It is howeveradvantageous to use a stabilization technology that is compatible withmost standard nucleic acid isolation methods and the use of silicacolumns for isolating extracellular nucleic acids is widely used andestablished. Using the high molecular weight poly(oxyethylene) polymerin a concentration of 1.5% (w/v) or less, 1.25% (w/v) or less, 1% (w/v)or less or 0.75% or less in the stabilization mixture containing thesample supports that the extracellular nucleic acids can be efficientlyisolated from the stabilized samples using such standard methods withgood yield even if higher volumes of stabilization composition is used.This is advantageous, because extracellular nucleic acids and inparticular specific target nucleic acids comprised in the extracellularnucleic acid populations are often present in only few copies. Thestabilization technology described herein is also compatible with othernucleic acid isolation methods, including those that are based on ionexchange. As is demonstrated in the examples, the observed impairmentalso depends on the used volume of the the stabilization compositionthat contains the high molecular weight (polyoxyethylene) polymer. Usinga lower volume of stabilizing composition for stabilization cancompensate the impairment even if a high molecular weightpoly(oxyethylene) polymer is used in higher concentrations so that thesubsequent nucleic acid isolation is not impaired. Thus, reducing theoverall concentration of the high molecular weight poly(oxyethylene)polymer in the mixture containing the sample and/or reducing the volumeof stabilsation composition containing the high molecular weightpoly(oxyethylene) polymer are alternative options to reduce or evenavoid impairment. This volume dependent effect that is demonstrated inthe examples is significant and was highly surprising as the overallconcentration in the mixture containing the sample was the same.

According to one embodiment, the poly(oxyethylene) polymer used forstabilization has a molecular weight below 1500 and may be a lowmolecular weight poly(oxyethylene) polymer having a molecular weight of1000 or less. It is used in a concentration, wherein it can exert orsupport the stabilizing effect on the extracellular nucleic acidpopulation of the cell-containing biological sample. Suitableconcentrations for different sample types can be determined by theskilled person, e.g. by testing different concentrations in the testassays described in the examples. A respective poly(oxyethylene)polymer, such as a low molecular weight poly(oxyethylene) polymer havinga molecular weight of 1000 or less, can be present in the mixture thatis obtained after contacting the cell-containing biological sample withsaid poly(oxyethylene) polymer and optionally further additives in aconcentration range that is selected from 0.5% to 10%, 1.5% to 9%, 2% to8%, 2 to 7%, 2.5% to 7% and 3% to 6%. The percentage values refer to(w/v) in case the poly(oxyethylene) polymer is a solid and to (v/v) incase the poly(oxyethylene) polymer is a liquid. The indicatedconcentrations are particularly suitable for the use in case of bloodsamples. Higher concentration of at least 1%, preferably at least 1.5%are advantageous for a low molecular weight poly(oxyethylene) polymer toachieve (or support) the stabilization of the extracellular nucleic acidpopulation. As is demonstrated in the examples, also poly(oxyethylene)polymers such as polyethylene glycol having a molecular weight of 1500or less such as 1000 or less show a stabilizing effect on theextracellular nucleic acid population, in particular by reducingdilutuions with genomic DNA. The examples demonstrate thatpoly(oxyethylene) polymers such as polyethylene glycol having amolecular weight of 1500 or less, such as 1000 or less are effectivestabilizers, in particular when used in combination with one or morefurther stabilizing agents described herein such as a caspase inhibitorand at least one primary, secondary or tertiary amide. The low molecularweight poly(oxyethylene) polymer may have a molecular weight that liesin a range selected from 100 to 1000, 150 to 800, 150 to 700, preferably200 to 600 and more preferably 200 to 500 such as 200 to 400.

It was found that a low molecular weight poly(oxyethylene) polymerhaving a molecular weight of 1000 or less, preferably of 800 or 700 orless, can be used in substantially higher concentrations, because itdoes not substantially hinder the subsequent isolation of theextracellular nucleic acids from the stabilized sample even if a highervolume of the stabilizing composition is used. However, if the amountrequired to achieve a stabilization effect is too high, this can beinconvenient for the processing and handling of the samples. It isgenerally preferred to stabilize the sample with a rather low amount orvolume of stabilizing agents. This particularly, as in case of certainsamples such as blood samples, the amount of stabilizing agent that canbe added to the sample is restricted by the standard collection tubesthat are used. E.g. for a standard collection device that is used forcollecting 10 ml blood, approx. 2 ml stabilizing agent can be added asmaximum.

According to one embodiment, at least two poly(oxyethylene) polymers areused for stabilization, which differ in their molecular weight. They maybe of the same kind and preferably both are a polyethylene glycol suchas an unsubstituted polyethylene glycol. According to one embodiment,the difference in the molecular weight is at least 100, at least 200, atleast 300, at least 400, at least 500, at least 600, at least 700, atleast 800, at least 900 or at least 1000. According to one embodiment,the difference in the molecular weight is at least 2500, at least 3500,at least 5000 or at least 7500. As is described subsequently in detailfor specific embodiments of this embodiment, it is advantageous to usetwo poly(oxyethylene) polymers that differ in their molecular weight. Asis described herein, the stabilization effect of poly(oxyethylene)polymers appears to depend on their molecular weight. In the testedexamples it was found that the higher the molecular weight, the higherthe stabilization efficiency. However, poly(oxyethylene) polymers maydiffer in their effect on the subsequent nucleic acid isolation methoddepending on their molecular weight. As described above, it was foundthat in certain embodiments that involve the use of higher molecularweight poly(oxyethylene) polymers as stabilizing agents, in particularwhere a higher volume of stabilization composition and a higherconcentration of the polymer in the stabilizing mixture containing thesample was used, that the nucleic acid isolation was less efficient withcertain nucleic acid isolation methods. As is demonstrated by theexamples, such issues that occur in certain scenarios can be overcomewhen using a mixture of poly(oxyethylene) polymers that differ in theirmolecular weight. Therefore, this embodiment wherein at least twopoly(oxyethylene) polymers are used for stabilization that differ intheir molecular weight is advantageous, because it allows to providebalanced compositions of poly(oxyethylene) polymers having the desiredcharacteristics with respect to the stabilization effect to be achievedand the characteristics required e.g. for certain downstream uses.

According to one embodiment, a high molecular weight poly(oxyethylene)polymer as defined above which has a molecular weight of at least 1500is used in combination with a low molecular weight poly(oxyethylene)polymer having a molecular weight of 1000 or less and thecell-containing sample is contacted with both types of poly(oxyethylene)polymers. Suitable embodiments are described herein. Using a highmolecular weight poly(oxyethylene) polymer in combination with a lowmolecular weight poly(oxyethylene) polymer is advantageous, because thelow molecular weight poly(oxyethylene) polymer allows to reduce theconcentration of the high molecular weight poly(oxyethylene) polymerrequired to achieve an effective stabilization of the sample. Therefore,the high molecular weight poly(oxyethylene) polymer can be used in themixture with the sample in a concentration wherein it does not impairthe subsequent nucleic acid isolation using certain standard methodssuch as those involving silica columns. This embodiment is andvantageousbecause it provides more freedom with respect to the volume or amount ofstabilization composition that can be used. The low molecular weightpoly(oxyethylene) polymer assists in the stabilization but in contrastto the high molecular weight poly(oxyethylene) polymer, was in thetested examples not found to significantly impair the subsequentisolation of the nucleic acids when using methods, such as thoseinvolving silica columns, where the higher molecular weightpoly(oxyethylene) polymers showed in certain concentrations and/orvolumes an impairing effect in the examples. Therefore, stabilizing theextracellular nucleic acid population in a cell-containing sample usinga combination of a high and a low molecular weight poly(oxyethylene)polymer is a particular preferred embodiment that has importantadvantages. The low molecular weight poly(oxyethylene) polymer can be ofthe same kind as the high molecular weight poly(oxyethylene) polymerwhich was described above. Also for the low molecular weightpoly(oxyethylene) polymer it is preferred that a polyethylene glycol isused, such as an unsubstituted polyethylene glycol. Suitable molecularweights for the high molecular weight poly(oxyethylene) polymer weredescribed above. It may have e.g. a molecular weight that lies in arange selected from 1500 to 50000, 2000 to 40000, 2500 to 30000, 2500 to25000 and 3000 to 20000. The low molecular weight poly(oxyethylene)polymer may have a molecular weight that lies in a range selected from100 to 1000, 150 to 800, 150 to 700, preferably 200 to 600 and morepreferably 200 to 500 such as 200 to 400. Suitable and preferredconcentrations and concentration ranges for the high and low molecularweight poly(oxyethylene) polymer are described above and may also beused in the embodiment wherein both types of polymers are used incombination.

According to one embodiment, the mixture that is obtained aftercontacting the cell-containing biological sample, which preferably isblood, with the high and the low molecular weight poly(oxyethylene)polymer and optionally further additives used for stabilization,comprises the high molecular weight poly(oxyethylene) polymer, which mayhave e.g. a molecular weight in the range of 3000 to 40000, preferably4000 to 20000, in a concentration that lies in a range of 0.2% to 1.5%(w/v), preferably 0.3% to 1.25% (w/v) and in embodiments in a range of0.4 (w/v) to 0.75% (w/v) and the low molecular weight poly(oxyethylene)polymer, which preferably has a molecular weight that lies in a range of200 to 800, preferably 200 to 600, in a concentration that lies in arange selected from 1.5% to 8%, preferably 2% to 7%, more preferred 2.5%to 6%. The high and low poly(oxyethylene) polymer is preferably apolyethylene glycol, such as an unsubstituted polyethylene glycol.Suitable embodiments for the high and low molecular weightpoly(oxyethylene) polymer are also described above and in conjunctionwith the different aspects and embodiments. The cell-containingbiological sample may be blood in this embodiment and the blood sampleis additionally contacted with an anticoagulant. Suitable examples foranticoagulants are described below.

As is shown by the provided examples, using a poly(oxyethylene) polymer,such as a high molecular weight poly(oxyethylene) polymer, alone is inembodiments already effective in stabilizing a cell-containing sampleand preserving the extracellular nucleic acid population from changes inits composition, in particular changes arising from a contamination withfragmented genomic DNA released from damaged or dying cells duringstorage. The stabilization effect can be significantly improved thoughif the poly(oxyethylene) polymer which preferably is a polyethyleneglycol is used in combination with further stabilization agents and/oradditives. It was found that using a poly(oxyethylene) polymer incombination with further stabilizing agents siginificantly improves theachieved stabilization effect. As is demonstrated in the examples, it issignificantly supporting and improving the stabilizing effect when beingused in combination with other stabilizing agents such as amides and acaspase inhinitor. Such balanced compositions significantly improve theachieved stabilization effect which is also superior to existing priorart technologies. Suitable and preferred examples of additionalstabilizing agents and innovative combinations are describedsubsequently.

According to one embodiment, the cell-containing sample is additionallycontacted with one or more primary, secondary or tertiary amides asfurther stabilizing agent. Primary, secondary and tertiary amides havean advantageous stabilization effect on cell-containing samples andtherefore, are in one embodiment used to support the stabilization ofthe extracellular nucleic acid population. Also combinations or two ormore primary, secondary or tertiary amides can be used. The amidepreferably is a carboxylic acid amide.

Suitable concentrations for different primary, secondary or tertiaryamides and/or for different sample types can be determined by theskilled person, e.g. by testing different concentrations in the testassays described in the examples. Generally, the mixture that isobtained when contacting the cell-containing biological sample with theat least one poly(oxyethylene) polymer and the one or more primary,secondary or tertiary amides and optionally further additives maycomprise said amide (or combination of amides) in a concentration of atleast 0.05%, at least 0.1%, at least 0.25%, at least 0.5% or at least0.75%. Suitable concentration ranges include but are not limited to 0.1%to 10%, 0.25% to 7.5%, 0.3% to 5%, 0.4% to 3%, 0.5% to 2%, 0.6% to 1.8%and 0.75% to 1.5%.

Concentrations or concentration ranges indicated in percentage values asused herein are in particular given as percentage weight per volume(w/v) for solid amides and as percentage volume per volume (v/v) forliquid amides.

According to one embodiment, the primary, secondary or tertiary amide isa compound according to formula 1

wherein R1 is a hydrogen residue or an alkyl residue, preferably a C1-C5alkyl residue, a C1-C4 alkyl residue or a C1-C3 alkyl residue, morepreferred a C1-C2 alkyl residue, R2 and R3 are identical or differentand are selected from a hydrogen residue and a hydrocarbon residue,preferably an alkyl residue, with a length of the carbon chain of 1-20atoms arranged in a linear or branched manner, and R4 is an oxygen,sulphur or selenium residue, preferably R4 is oxygen.

Also a combination of one or more compounds according to formula 1 canbe used in addition to the poly(oxyethylene) polymer for stabilization.

In embodiments, wherein R1 is an alkyl residue, a chain length of 1 or 2is preferred for R1.

R2 and/or R3 of the compound according to formula 1 are identical ordifferent and are selected from a hydrogen residue and a hydrocarbonresidue, which preferably is an alkyl residue. According to oneembodiment, R2 and R3 are both hydrogen. According to one embodiment,one of R2 and R3 is a hydrogen and the other is a hydrocarbon residue.According to one embodiment, R2 and R3 are identical or differenthydrocarbon residues. The hydrocarbon residues R2 and/or R3 can beselected independently of one another from the group comprising alkyl,including short chain alkyl and long-chain alkyl, alkenyl, alkoxy,long-chain alkoxy, cycloalkyl, aryl, haloalkyl, alkylsilyl,alkylsilyloxy, alkylene, alkenediyl, arylene, carboxylates and carbonyl.The chain length n of R2 and/or R3 can in particular have the values 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20.According to one embodiment, R2 and R3 have a length of the carbon chainof 1-10, preferably 1 to 5, more preferred 1 to 2. According to oneembodiment, R2 and/or R3 are alkyl residues, preferably C1-C5 alkylresidues.

Preferably, the compound according to formula 1 is a carboxylic acidamide so that R4 is oxygen. It can be a primary, secondary or tertiarycarboxylic acid amide.

According to one embodiment, the compound according to formula 1 is aN,N-dialkyl-carboxylic acid amide. Preferred R1, R2, R3 and R4 groupsare described above. Using a respective compound according to formula 1has the advantage that additionally, intracellular nucleic acids such asin particular RNA, e.g. mRNA and/or miRNA transcripts can be stabilizedin the cell-containing sample. The additional stabilization ofintracellular nucleic acids, in particular gene transcript levels, isadvantageous as it e.g. allows the subsequent analysis of targettranscripts or transcript profiles in the contained cells. According toone embodiment, the compound according to formula 1 is selected from thegroup consisting of N,N-dimethylacetamide, N,N-diethylacetamide,N,N-dimethylformamide and N,N-diethylformamide. Also suitable are therespective thio analogues, which comprise sulphur instead of oxygen asR4. N,N-dimethlylacetamide (DMAA) e.g. achieves good stabilizationresults, however, is a toxic agent. Preferably, at least one compoundaccording to formula 1 is used in combination with the poly(oxyethylene)polymer for stabilizing the cell-containing biological sample which isnot a toxic agent according to the GHS classification.

According to one embodiment, the cell-containing biological sample iscontacted for stabilization with a poly(oxyethylene) polymer and acompound according to formula 1 which is a N,N-dialkylpropanamide, suchas N,N-dimethylpropanamide. As is demonstrated in the examples, arespective combination is particularly suitable for stabilizing theextracellular nucleic acid population in a cell-containing sample suchas a blood sample.

According to one embodiment, the cell-containing sample is contactedwith a poly(oxyethylene) polymer and at least one compound according toformula 1, which is a primary or secondary carboxylic acid amide. As canbe seen from the examples of unpublished U.S. 61/803,107 and EP 13 160896.0 (see published WO 2014/146781), herein incorporated by reference,primary and secondary carboxylic acid amides are capable of stabilizingthe extracellular nucleic acid population over a broad concentrationrange. According to one embodiment, the primary carboxylic acid amide isselected from the group consisting of formamide, acetamide, propanamideand butanamide. Preferably, the primary carboxylic acid is butanamide,as butanamide is particularly effective for stabilizing theextracellular nucleic acid population as demonstrated in the presentexamples and additionally, is non-toxic. The stabilization effect ofbutanamide on the extracellular nucleic acid population ofcell-containing samples is also described in detail in unpublishedapplications EP 13 159 834.4 and EP 13 180 086.4 (see published WO2014/146780 and WO 2014/146782), herein incorporated by reference.

According to a preferred embodiment, the cell-containing sample iscontacted for stabilization with the poly(oxyethylene) polymer, whichpreferably is a polyethylene glycol, and butanamide and/or anN,N-dialkylpropanamide, wherein said N,N-dialkylpropanamide preferablyis N,N-dimethylpropanamide. Suitable and preferred concentrations andconcentration ranges described above in general for the primary,secondary and tertiary amides in general also specifically apply to thisembodiment. As is shown by the examples, using butanamide and/or aN,N-dialkylpropanamide in a concentration that lies in these rangesprovides an advantageous stabilizing effect on cell-containing samplessuch as blood samples.

According to one embodiment, the cell-containing biological sample iscontacted with a poly(oxyethylene) polymer as described above and atleast one caspase inhibitor. It is also within the scope of the presentinvention to use a combination of different caspase inhibitors. As isshown by the examples, using a poly(oxyethylene) polymer such aspreferably a high molecular weight polyethylene glycol in combinationwith a caspase inhibitor significantly improves the achievedstabilization effect. Furthermore, it was found that in particular withbiological samples that contain large amounts of cells and furthermore,differ in their composition, such as e.g. blood samples, the achievedstabilization effect was stronger and more uniform. E.g. blood samplesderived from different donors may differ in the changes in theextracellular nucleic acid population that occur during ex vivohandling. Some samples show strong alterations in the profile of theextracellular nucleic acid population (in particular strong increases ingenomic DNA) while in other samples the effects are less prominent. Suchsamples can also react differently to stabilization. When using apoly(oxyethylene) polymer in combination with a caspase inhibitor andpreferably also one or more primary, secondary or tertiary amide asdescribed above for stabilizing blood samples obtained from differentdonors, a more uniform stabilization effect could be achieved. Thecaspase inhibitor present in the resulting mixture significantlysupports the stabilization of the extracellular nucleic acid population.Furthermore, the degradation of nucleic acids, in particular genomicDNA, present in the sample is reduced by said combination of stabilizingagents. Thus, using a poly(oxyethylene) polymer such as polyethyleneglycol in combination with at least one caspase inhibitor significantlyimproves the stabilization effect thereby supporting that theextracellular nucleic acid population contained in the sample issubstantially preserved in the state it had shown at the time thebiological sample was obtained, respectively collected, even duringprolonged storage periods.

Preferably, the caspase inhibitor is cell-permeable. Members of thecaspase gene family play a significant role in apoptosis. The substratepreferences or specificities of individual caspases have been exploitedfor the development of peptides that successfully compete caspasebinding. It is possible to generate reversible or irreversibleinhibitors of caspase activation by coupling caspase-specific peptidesto e.g. aldehyde, nitrile or ketone compounds. E.g. fluoromethyl ketone(FMK) derivatized peptides such as Z-VAD-FMK act as effectiveirreversible inhibitors with no added cytotoxic effects. Inhibitorssynthesized with a benzyloxycarbonyl group (BOC) at the N-terminus andO-methyl side chains exhibit enhanced cellular permability. Furthersuitable caspase inhibitors are synthesized with a phenoxy group at theC-terminus. An example is Q-VD-OPh which is a cell permeable,irreversible broad-spectrum caspase inhibitor that is even moreeffective in preventing apoptosis and thus supporting the stabilizationthan the caspase inhibitor Z-VAD-FMK.

According to one embodiment, the caspase inhibitor is a pancaspaseinhibitor and thus is a broad spectrum caspase inhibitor. According toone embodiment, the caspase inhibitor comprises a modifiedcaspase-specific peptide. Preferably, said caspase-specific peptide ismodified by an aldehyde, nitrile or ketone compound. According to oneembodiment, the caspase specific peptide is modified, preferably at thecarboxyl terminus, with an O-Phenoxy (OPh) or a fluoromethyl ketone(FMK) group. According to one embodiment, the caspase inhibitor isselected from the group consisting of Q-VD-OPh and Z-VAD(OMe)-FMK. In apreferred embodiment, Q-VD-OPh, which is a broad spectrum inhibitor forcaspases, is used for stabilization. Q-VD-OPh is cell permeable andinhibits cell death by apoptosis. Q-VD-OPh is not toxic to cells even atextremely high concentrations and comprises a carboxy terminal phenoxygroup conjugated to the amino acids valine and aspartate. It is equallyeffective in preventing apoptosis mediated by the three major apoptoticpathways, caspase-9 and caspase-3, caspase-8 and caspase-10, andcaspase-12 (Caserta et al, 2003). Examples of caspase inhibitors arealso listed in Table 1 of WO 2013/045457, herein incorporated byreference.

The mixture that is obtained after contacting the biological sample withthe at least one poly(oxyethylene) polymer and the at least one caspaseinhibitor and optionally further additives may comprise the caspaseinhibitor (or combination of caspase inhibitors) in a concentration ofat least at least 0.05 μM, at least 0.1 μM, at least 0.5 μM, at least0.75 μM, at least 1 μM, at least 1.25 μM, at least 1.5 μM, at least 1.75μM, at least 2 μM, at least 2.25 μM, at 2.5 μM, at least 2.75 μM, atleast 3 μM, at least 3.25 μM or at least 3.5 μM. Suitable concentrationranges for the caspase inhibitor(s) when mixed with the cell-containingbiological sample and the further additives include but are not limited0.1 μM to 25 μM, 0.75 μM to 20 μM, 1 μM to 15 μM, 1.5 μM to 12.5 μM, and2 μM to 10 μM and 3 μM to 7.5 μM. The above mentioned concentrationsapply to the use of a single caspase inhibitor as well as to the use ofa combination of caspase inhibitors. The aforementioned concentrationsare in particular suitable when using a pancaspase inhibitor, inparticular a modified caspase specific peptide such as Q-VD-OPh and/orZ-VAD(OMe)-FMK. The above mentioned concentrations are e.g. suitable forstabilizing whole blood. Suitable concentration ranges for individualcaspase inhibitors and/or for other cell-containing biological samplescan be determined by the skilled person, e.g. by testing differentconcentrations of the respective caspase inhibitor in the test assaysdescribed in the examples.

The stabilizing effect observed with combinations of stabilizing agentsis stronger than the effect observed for any of the individualstabilizing agents when used alone and/or allows using lowerconcentrations of individual stabilizers, thereby making combinatorialuse of stabilizing agents an attractive option. The combinations ofstabilizing agents involving a poly(oxyethylene) polymer such as apolyethylene glycol described herein have a better stabilizing effectthan the individual stabilizers and therefore, are advantageousembodiments. Furthermore, additional additives can be used forstabilization such as e.g. anticoagulants and chelating agents which areparticularly useful when stabilizing a blood sample.

As discussed in the background of the invention, extracellular nucleicacids are usually not present “naked” in the extracellular portion ofthe cell-containing sample but are e.g. stabilized to a certain extentby being released protected in complexes or by being contained invesicles and the like. This has the effect that extracellular nucleicacids are already to a certain extent stabilized by nature and thus, areusually not degraded rapidly by nucleases in cell-containing samplessuch as whole blood, plasma or serum. Thus, when intending to stabilizeextracellular nucleic acids that are comprised in a cell-containingbiological sample, one of the primary problems after obtaining orcollecting the cell-containing biological sample is the contaminationand dilution of the extracellular nucleic acid population comprised inthe collected cell-containing biological sample by intracellular nucleicacids, in particular fragmented genomic DNA, that originates fromdamaged or dying cells that are contained in the cell-containingbiological sample. The stabilization technology according to the presentinvention is of particular advantage in this respect because it not onlysubstantially preserves the extracellular nucleic acids present in thesample and e.g. inhibits degradation of the comprised extracellularnucleic acids (preferably at least by 60%, at least by 70%, at least by75%, at least by 80%, at least by 85%, at least by 90% or mostpreferably at least by 95% over the stabilization period compared to anunstabilized sample or e.g. an EDTA stabilized sample in the case ofblood) but furthermore, efficiently reduces the release of genomic DNAfrom cells contained in the obtained cell-containing biological sampleand/or reduces the fragmentation of respective genomic DNA. According toone embodiment, using a poly(oxyethylene) polymer for stabilizing theextracellular nucleic acid population in a cell-containing sample,optionally but preferably in combination with one or more of the furtherstabilization agents described above, has the effect that the increaseof DNA that results from a release of genomic DNA from cells containedin the sample during the stabilization period is reduced compared to anon-stabilized sample. According to one embodiment, said release ofgenomic DNA is reduced by at least 3-fold, at least 4-fold, at least5-fold, at least 6-fold, at least 7-fold, at least 10-fold, at least12-fold, at least 15-fold, at least 17-fold or at least 20-fold over thestabilization period compared to the non-stabilized sample or acorresponding sample that is stabilized with EDTA (in particular in caseof a blood sample or a sample derived from blood such as plasma orserum). According to one embodiment, said release of genomic DNA isreduced by at least 60%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90% or at least 95% over the stabilization periodcompared to the non-stabilized sample or a corresponding sample that isstabilized with EDTA (in particular in case of a blood sample or asample derived from blood such as plasma or serum). The release of DNAcan be determined e.g. by quantifying the ribosomal 18S DNA as isdescribed herein in the example section. As is shown in the examples,the stabilization achievable with the teachings of the present inventionremarkably reduces this release of DNA over the stabilization period.Thus, according to one embodiment, the stabilization effect that isachieved with a poly(oxyethylene) polymer such as a polyethylene glycol,optionally in combination with one or more of the additionalstabilization agents described above such as preferably one or moreprimary, secondary or tertiary amides and a caspase inhibitor, resultsin that the release of DNA from cells contained in the stabilized sampleis over the stabilization period reduced at least down to a maximum of8-fold, at least down to a maximum of 7-fold, at least down to a maximumof 5-fold, more preferably is reduced e.g. at least down to a maximum of4-fold, more preferably is reduced at least down to a maximum of 3-foldor preferably down to a maximum of 2-fold or even less as is e.g.determinable in the 18S DNA assay described in the examples. As is shownby the examples, an effective stabilization of the extracellular nucleicacid population is achievable with the method of the invention for aperiod of at least three days and when using a combination ofstabilizing agents as described herein even up to 6 days and longer.During shorter as well as during longer storage of the stabilizedsamples the DNA release can be reduced in embodiments at least down to amaximum of two-fold and lower as is e.g. determinable in the 18S DNAassay described in the examples. Thus, according to embodiments of thestabilization technology described herein, the DNA release can bereduced down to 3 fold or less or even 2 fold or less up to three oreven up to 6 days of storage and longer when using the stabilizingmethods according to the present invention. As is demonstrated by theexamples, in embodiments values between 1 and 1.5 are achieved. This isa remarkable improvement in the stabilization of the extracellularnucleic acid population compared to prior art methods. However, ofcourse, the samples may also be further processed earlier, if desired.It is not necessary to make use of the full achievable stabilizationperiod. Furthermore, nucleic acids can be efficiently isolated fromrespectively stabilized samples using different standard methods as nocross-linking of the sample occurs due to the stabilization. Thisgreatly simplifies and improves the standardization of molecularanalysis that relies on the analysis of extracellular nucleic acids.

The selection of suitable additives that may also contribute to thestabilization effect may also depend on the type of cell-containingsample to be stabilized. E.g. when processing blood as cell-containingbiological sample, an anticoagulant is additionally used to preventblood clotting. The anticoagulant is used in a concentration wherein itcan prevent clotting of the amount of blood to be stabilized. Theanticoagulant may be e.g. selected from the group consisting of heparin,chelating agents such as ethylenediamine tetraacetic acid, salts ofcarboxylic acids such as citrate or oxalate and any combination thereof.In an advantageous embodiment, the anticoagulant is a chelating agent. Achelating agent is an organic compound that is capable of formingcoordinate bonds with metals through two or more atoms of the organiccompound. Chelating agents according to the present invention include,but are not limited to diethylenetriaminepentaacetic acid (DTPA),ethylenedinitrilotetraacetic acid (EDTA), ethylene glycol tetraaceticacid (EGTA) and N,N-bis(carboxymethyl)glycine (NTA) and furthermore,e.g. citrate or oxalate. According to a preferred embodiment, EDTA isused as anticoagulant. As used herein, the term “EDTA” indicates interalia the EDTA portion of an EDTA compound such as, for example, K₂EDTA,K₃EDTA or Na₂EDTA. Using a chelating agent such as EDTA also has theadvantageous effect that nucleases such as DNases and RNases areinhibited, thereby e.g. preventing a degradation of extracellularnucleic acids by nucleases. Therefore, the use of a chelating agent suchas EDTA is also advantageous when using cell-containing samplesdifferent from blood. Furthermore, it was found that EDTA used/added inhigher concentrations supports the stabilizing effect. However, EDTAalone does not achieve a sufficient stabilization effect for thepurposes described herein. However, used in combination with theteachings of the present invention, in particular in combination with apoly(oxyethylene) polymer (and one or more of the further stabilizingagents as described herein, such as preferably a primary, secondary ortertiary amide and/or a caspase inhibitor), it can further improve thestabilization effect for the above discussed reasons.

According to one embodiment, the concentration of the chelating agent,preferably EDTA, in the mixture that is obtained when contacting thecell-containing biological sample with the poly(oxyethylene) polymer andoptionally one or more additional additives lies in a range selectedfrom the group consisting of 0.5 to 40 mg/ml, 1 to 30 mg/ml, 1.6 to 25mg/ml, 5 to 20 mg/ml and 7.5 to 17.5 mg/ml. Respective concentrationsare particularly effective when stabilizing blood, plasma and/or serumsamples. Suitable concentrations can also be determined by the skilledperson.

Additional additives can also be used in order to further support thestabilization of the cell-containing sample, respectively support thepreservation of the extracellular nucleic acid population. Examples ofrespective additives include but are not limited to nuclease inhibitors,in particular RNase and DNase inhibiting compounds. When choosing arespective further additive for supporting stabilization, care should betaken not to compromise and/or counteract the stabilizing effect. Thus,no additives such as e.g. chaotropic agents should be used inconcentrations that result in or support the lysis and/or degradation ofnucleated cells contained in the cell-containing biological sample thatis stabilized and/or which support the degradation of the nucleic acidscontained in the cell-free fraction of said biological sample.Therefore, preferably, the stabilization method described herein doesnot involve the use of additives (i) that induce or promote lysis ofnucleated cells, (ii) that induce or promote lysis of cells in generaland/or (iii) that lead to a degradation of nucleic acids contained inthe cell-free fraction of the cell-containing biological sample. As thestabilization method described herein is not based on cell lysis butpreserve cells, cells can be separated from the cell-containing sampleafter the stabilization period, thereby allowing to obtain a cell-freeor cell-depleted fraction which comprises the extracellular nucleic acidpopulation. Due to the poly(oxyethylene) polymer based stabilizationdescribed herein, said extracellular nucleic acid populationsubstantially corresponds to or at least closely resembles theextracellular nucleic acid population present at the time of samplecollection and stabilization. Furthermore, nucleic acids can be isolatedfrom the separated cells and are available for analysis. As describedabove, a combination comprising a compound according to formula 1 alsohas transcriptome stabilizing properties. By stabilizing thetranscriptome in addition to the extracellular nucleic acid population,the respectively stabilized samples are also suitable for geneexpression profiling. Furthermore, respectively stabilized samplesallow, if desired, the separate analysis of the extracellular andintracellular nucleic acid populations from the same stabilized sample.

In an advantageous embodiment, the cell-containing biological sample,which preferably is a blood sample or a sample derived from blood suchas plasma or serum, is contacted with:

-   -   a) at least one high molecular weight poly(oxyethylene) polymer        having a molecular weight of at least 1500, preferably in a        range of 1500 to 50000, 2000 to 40000, 2500 to 30000, 2500 to        25000, more preferred 3000 to 20000 or 4500 to 10000;    -   b) one or more compounds according to formula 1, preferably in a        concentration so that the concentration in the mixture with the        cell-containing biological sample lies in a range of 0.25% to        5%, 0.3% to 4%, 0.4% to 3%, 0.5% to 2% or 0.75% to 1.5%;    -   c) at least one caspase inhibitor, preferably a pancaspase        inhibitor, more preferred Q-VD-OPh, preferably in a        concentration so that the concentration of the caspase inhibitor        in the mixture with the cell-containing biological sample lies        in a range of 0.1 μM to 20 μM, more preferred 0.5 μM to 10 μM,        more preferred 1 μM to 10 μM, more preferred 3 μM to 7.5 μM or 3        μM to 5 μM;    -   d) optionally at least one further poly(oxyethylene) polymer        having a molecular weight that is at least 100, preferably at        least 200, at least 300 or at least 400 below the molecular        weight of the high molecular weight poly(oxyethylene) polymer        used and wherein said further poly(oxyethylene) polymer        preferably is a low molecular weight poly(oxyethylene) having a        molecular weight of 1000 or less, preferably having a molecular        weight in a range of 200 to 800 or 200 to 600;    -   e) optionally a chelating agent, more preferably EDTA.

In an advantageous embodiment, the cell-containing biological sample,which preferably is a blood sample or a sample derived from blood suchas plasma or serum, is contacted with:

-   -   a) at least one high molecular weight poly(oxyethylene) polymer,        preferably polyethylene glycol, having a molecular weight in a        range of 2000 to 40000, 2500 to 30000, 3000 to 25000, 3500 to        20000 or 4000 to 15000;    -   b) one or more compounds according to formula 1, preferably        butanamide and/or an N,N-dialkyl propanamide;    -   c) at least one caspase inhibitor, preferably a pancaspase        inhibitor, more preferred 0-VD-OPh;    -   d) at least one low molecular weight poly(oxyethylene),        preferably polyethylene glycol, having a molecular weight in a        range of 100 to 1000, 150 to 800 or 200 to 600;

e) optionally a chelating agent, more preferably EDTA,

wherein after the cell-containing biological sample has been contactedwith said additives and optionally further additives used forstabilization the resulting mixture comprises

-   -   the high molecular weight poly(oxyethylene) polymer in a        concentration that lies in a range of 0.25% to 1.25% (w/v), 0.3%        to 1% (w/v) or 0.4% to 0.75% (w/v),    -   the one or more compounds according to formula 1 in a        concentration that lies in a range of 0.3% to 4%, 0.4 to 3% or        0.5 to 2%,    -   the caspase inhibitor in a concentration that lies in a range of        1 μM to 10 μM, preferably 3 μM to 7.5 μM, and    -   the low molecular weight poly(oxyethylene) polymer in a        concentration that lies in the range of 1.5% to 10%, 2% to 8%,        2.5 to 7% and 3% to 6%.

According to one embodiment, the method according to the presentinvention is for stabilizing an extracellular nucleic acid populationcomprised in a blood sample and comprises contacting the blood samplewith at least one poly(oxyethylene) polymer, preferably a high molecularweight poly(oxyethylene) polymer, preferably a polyethylene glycol, andan anticoagulant, wherein during the stabilization period, the releaseof genomic DNA from cells contained in the blood sample into thecell-free portion of the blood sample is reduced. In particular, thepresent invention provides a method for stabilizing an extracellularnucleic acid population comprised in a blood sample which comprisescontacting the blood sample with at least one poly(oxyethylene) polymer,one or more primary, secondary or tertiary amides (suitable andpreferred examples and concentrations are described above), at least onecaspase inhibitor and an anticoagulant and wherein the release ofgenomic DNA from nucleated cells contained in the blood sample into thecell-free portion of the blood sample is reduced. In particular, lysisof white blood cells is prevented/reduced during stabilization.Furthermore, degradation of nucleic acids present in the sample isreduced due to the stabilization.

In one embodiment, the cell-containing biological sample is a bloodsample which is contacted with:

-   -   a) at least one high molecular weight poly(oxyethylene) polymer        having a molecular weight that lies in a range of 2000 to 40000,        2500 to 30000, 2500 to 25000, 3000 to 20000, 3500 to 15000 or        3000 to 10000;    -   b) one or more compounds according to formula 1;    -   c) at least one caspase inhibitor, preferably a pancaspase        inhibitor, more preferred Q-VD-OPh;    -   d) at least one low molecular weight poly(oxyethylene) polymer        having a molecular weight of 1000 or less, preferably in a range        of 100 to 800, 200 to 600 or 200 to 500;    -   e) an anticoagulant which preferably is a chelating agent, more        preferably EDTA,        wherein after the blood sample has been contacted with said        additives and optionally further additives used for        stabilization the resulting mixture comprises    -   the high molecular weight poly(oxyethylene) polymer in a        concentration that lies in a range of 0.2% to 1.5% (w/v), 0.25%        to 1.25% (w/v), 0.3% to 1% (w/v) or 0.4% to 0.75% (w/v),    -   the one or more compounds according to formula 1 in a        concentration that lies in a range of 0.3% to 4%, preferably 0.5        to 3%, more preferred 0.5% to 2% or 0.75% to 1.5%,    -   the caspase inhibitor in a concentration that lies in a range of        1 μM to 10 μM, preferably 3 μM to 7.5 μM, and    -   the low molecular weight poly(oxyethylene) polymer in a        concentration that lies in the range of 1.5% to 10%, preferably        2% to 6%.

According to one embodiment, the method is for stabilizing anextracellular nucleic acid population comprised in a blood sample andcomprises contacting the blood sample with at least onepoly(oxyethylene) polymer, butanamide and/or an N,N-dialkylpropanamidesuch as N,N-dimethylpropanamide, at least one caspase inhibitor, and ananticoagulant, wherein the release of genomic DNA from cells containedin the blood sample into the cell-free portion of the blood sample isreduced. Preferably, said stabilization effect is achieved for at leastthree days, more preferred up to 6 days and longer. As described above,the release of DNA from the contained cells throughout the stabilizationperiod preferably does not exceed a maximum of 2-fold, preferably iseven approx. 1.5-fold or less.

In embodiments, a further advantage when stabilizing blood samples usingthe method according to the present invention is that hemolysis can besignificantly reduced during the stabilization period. In oneembodiment, the poly(oxyethylene) polymer and the one or more additionalagents used for stabilization are comprised in a stabilizationcomposition that contains water. The examples demonstrate that thisembodiment signicficantly reduces the hemolysis of the red blood cellscompared to unstabilized samples or standard EDTA blood samples and evencompared to other stabilized samples. Hemolysis is the rupturing oferythrocytes and the release of their cytoplasm into surroundingextracellular fluid, e.g. blood plasma. The degree of hemolysis can beanalysed e.g. by visual inspection, as the released hemoglobin willcause the serum or plasma to appear red. Most causes of in vitrohemolysis are related to specimen collection. However, in vitrohemolysis usually also occurs in a blood sample during ex vivo storageif no proper stabilization method is used. Depending on theextracellular nucleic acid of interest, hemolysis can be a considerableproblem. If the extracellular nucleic acid of interest is DNA, hemolysisis less of a problem because red blood cells do not contain a nucleusand consequently, do not contain genomic DNA. Therefore, nointracellular DNA is released from the red blood cells during hemolysis.When the extracellular nucleic acid of interest is DNA, in particularthe lysis or decay of white blood cells is a problem because in thiscase genomic DNA is released in addition to intracellular RNA.Therefore, when the extracellular nucleic acid of interest isextracellular DNA, in particular the lysis of white blood cells must beavoided. White blood cells may differ among each other in theirstability characteristics. Thus, some types of white blood cells aremore stable than others. However, generally, white blood cells aresignificantly more stable than red blood cells. Therefore, the lysis ofred blood cells does not necessarily indicate that white blood cellswere lysed. The different susceptibility of white blood cells and redblood cells to lysis is also used in the art to e.g. specifically lysered blood cells, while preserving white cells in order to allow e. g.the collection of white blood cells. However, if the extracellularnucleic acid of interest is RNA, hemolysis and thus the lysis of redblood cells does constitute a problem. Mature red blood cells also donot contain RNA, however, their precursors (reticulocytes) do.Reticulocytes make up approximately 0.5% to 1% of the red blood cellsand contain large amounts of globin RNA. Therefore, in particular whenthe extracellular nucleic acid of interest is RNA, it is advantageous toreduce or prevent lysis of red blood cells and thus reticulocytes duringstorage in order to reduce a dilution of the extracellular nucleic acidpopulation, in particular the extracellular RNA population, with globinmRNA. Furthermore, as described above, it is advantageous to maintainthe composition and thus profile of the extracellular nucleic acidpopulation. As is shown by the examples, in embodiments, hemolysis isefficiently reduced when using the stabilization method according to thepresent invention. This is particularly the case where a stabilizationcomposition comprising water is used. Thereby, the extracellular nucleicacid population is substantially preserved and furthermore, thestabilized blood sample, in particular the plasma or serum obtained fromthe stabilized blood sample, is due to the prevention of hemolysis andcell lysis in general also suitable for other standard laboratoryanalyses.

The poly(oxyethylene) polymer may be comprised in a stabilizingcomposition. If one or more further stabilizing agents and additives areadditionally used, they may and preferably are also be comprised in thestabilization composition. Preferably, a stabilizing compositionaccording to the third aspect of the present invention is used forstabilization of the cell-containing biological sample.

Furthermore, as described according to one alternative of the firstaspect, the cell-containing biological sample to be stabilized iscontacted with mono-ethylene glycol (1,2-ethanediol) as stabilizingagent. Mono-ethylene glycol may be used in the concentrations describedabove for the low molecular weight poly(oxyethylene) polymer. It isreferred to the respective disclosure which also applies here. Inparticular, the cell-containing biological sample to be stabilized maybe contacted with mono-ethylene glycol and any one or more of the otherstabilizing agents described herein, preferably with at least onecaspase inhibitor and/or with at least one primary, secondary ortertiary amide, which preferably is a compound according to formula 1 asdefined above. Mono-ethylene glycol may be used in combination with theat least one poly(oxyethylene) polymer. Suitable embodiments andconcentration ranges for the respective stabilizing agents are describedabove and also apply to the embodiment, wherein mono-ethylene glycol isused in order to stabilize or support the stabilization of acell-containing sample and the exracellular nucleic acid populationcomprised therein.

The components of the stabilizing composition can be comprised,respectively can be dissolved in a solvent, e.g. water, a buffer, e.g. abiological buffer such as MOPS, TRIS, PBS and the like. Furthermore, thecomponents may be dissolved in or the stabilizing composition maycomprise a polar aprotic solvent such as dimethyl sulfoxide (DMSO). Asis demonstrated by the examples, using a stabilization composition thatcontains water is particularly preferred when stabilizing a bloodsample, as this embodiment significantly reduces hemolysis.

The poly(oxyethylene) polymer and the optionally used furtherstabilizing agents and/or additives can be present in a device,preferably a container, for collecting the cell-containing biologicalsample. The poly(oxyethylene) polymer and optionally the one or morecompounds additionally used for stabilization can be present in astabilizing composition that is present in a respective device or can bepresent as separate entities. Furthermore, they can be added to arespective collection device prior to collection of the cell-containingbiological sample, or can be added to the collection device after thecell-containing biological sample was collected therein. It is alsowithin the scope of the present invention to add the stabilizing agentsand optionally, further additive(s) used separately to thecell-containing biological sample. However, for the ease of handling, itis preferred that the poly(oxyethylene) polymer and any furtherstabilizing agents and/or additives used are provided in the respectivecollection device, e.g. in form of a single composition. However, theymay also be present as separate components or compositions in thecollection device. In an advantageous embodiment, the at least onepoly(oxyethylene) polymer, the one or more primary, secondary ortertiary amides, the at least one caspase inhibitor, and optionallyfurther additive(s) such as e.g. an anticoagulant such as EDTA, arepresent in the collection device prior to adding the cell-containingbiological sample. This ensures that the cell-containing biologicalsample is rapidly stabilized upon contact with the stabilizing agentsused according to the teachings of the present invention. Thestabilization agents are present in the container in an amount effectiveto stabilize the amount of cell-containing biological sample to becollected, respectively contained in said collection device. Suitableand preferred embodiments for a respective collection device are alsodescribed subsequently in conjunction with the fifth embodiment and itis referred to said disclosure. The same applies when usingmono-ethylene glycol as stabilizing agent.

Preferably, the cell-containing biological sample is contacted with thepoly(oxyethylene) polymer and optionally further additives directlyafter and/or during the collection of the cell-containing biologicalsample. Therefore, as described above, preferably, the agents used forstabilization are provided in form of a stabilizing composition.Preferably, said stabilizing composition is provided in a liquid form.It can be e.g. pre-filled in the sample collection device so that thecell-containing biological sample is rapidly stabilized duringcollection. According to one embodiment, the stabilizing composition iscontacted with the cell-containing sample in a volumetric ratio selectedfrom 10:1 to 1:20, 5:1 to 1:15, 1:1 to 1:10, 1:10 to 1:5 and 1:7 to 1:5,in particular about 1:6. These ratios are particularly useful forstabilizing blood samples. For stabilizing blood samples it is preferredthat the stabilization composition contains water. Suitable andpreferred concentrations of the additives in the resulting mixture withthe cell-containing sample, in particular a blood sample, were describedabove and it is referred to the respective disclosure. It is aparticular advantage of teachings of the present invention thatstabilization of a large sample volume can be achieved with a smallvolume of the stabilizing composition according to the presentinvention. This particularly, if a high molecular weightpoly(oxyethylene) polymer as described above is used as this allows touse lower concentrations compared to a low molecular weightpoly(oxyethylene) polymer. Therefore, preferably, the ratio ofstabilizing composition to sample lies in a range from 1:10 to 1:5, inparticular 1:7 to 1:5, such as about 1.6.

The term “cell-containing biological sample”, “cell-containing sample”and similar terms as used herein, in particular refers to a sample whichcomprises at least 50, 250, at least 500, at least 1000, at least 1500,at least 2000 or at least 5000 cells. According to one embodiment, thecellular portion makes up at least 1%, at least 2%, at least 2.5%, atleast 5%, preferably at least 10%, at least 15%, at least 20%, morepreferably at least 25%, at least 30%, at least 35% or at least 40% ofthe cell-containing biological sample. Cell-containing samplescomprising considerably more cells, wherein the cellular fraction makesup more than 40% can also be stabilized using the teachings describedherein. However, the term “cell-containing biological sample” alsorefers to and thus encompasses cell-depleted samples, includingcell-depleted samples that are commonly referred to as “cell-free” suchas e.g. blood plasma as respective samples often include residual cells.At least, it can often not be fully excluded that even so-called“cell-free” samples such as blood plasma comprise residual amounts ofcells which accordingly, pose a risk that the extracellular nucleic acidpopulation becomes contaminated with intracellular nucleic acidsreleased from said residual cells. Therefore, respective cell-depletedand “cell-free” samples are according to one embodiment also encompassedby the term “cell-containing biological sample”. Thus, the“cell-containing sample” may comprise large amounts of cells, as is thecase e.g. with whole blood, but may also only comprise merely minoramounts of cells. Hence, the term “cell containing biological sample”also encompasses samples that may only be suspected of or pose a risk ofcontaining cells. As discussed above, also with respect to biologicalsamples which only comprise minor, respectively residual amounts ofcells such as e.g. blood plasma (blood plasma contains depending on thepreparation method usually small residual amounts of cells, even thoughit is commonly referred to as being cell-free), the method according tothe present invention has considerable advantages as these residualcells may also result in a undesired contamination of the comprisedextracellular nucleic acids. Using the stabilizing method according tothe present invention has the advantage that substantially irrespectiveof the composition of the cell-containing biological sample and thenumber of cells contained therein, the extracellular nucleic acidpopulation contained in said sample can be substantially preserved,respectively stabilized, thereby allowing for standardizing thesubsequent isolation and/or analysis of the contained extracellularnucleic acids.

According to one embodiment, the cell-containing biological sample isselected from the group consisting of body fluids and cell-containingsamples derived from body fluids, in particular, whole blood, samplesderived from blood such as plasma or serum, buffy coat, urine, sputum,lachrymal fluid, lymphatic fluid, sweat, liquor, cerebrospinal fluid,ascites, milk, stool, bronchial lavage, saliva, amniotic fluid, nasalsecretions, vaginal secretions, semen/seminal fluid, wound secretions,cell culture and swab samples. According to one embodiment, thecell-containing biological sample is a body fluid, a body secretion orbody excretion, preferably a body fluid, most preferably urine,lymphatic fluid, blood, buffy coat, plasma or serum. In particular, thecell-containing biological sample can be a circulating body fluid suchas blood or lymphatic fluid. Preferably, the cell-containing biologicalsample that is stabilized using the teachings described herein is ablood sample, sometimes also referred to whole blood. The blood samplepreferably has not been diluted or fractionated prior to stabilization.According to one embodiment, the blood sample is peripheral blood.According to one embodiment, the cell-containing biological sample wasobtained from a human. The cell-containing biological sample comprisesextracellular nucleic acids in the extracellular portion.

The term “extracellular nucleic acids” or “extracellular nucleic acid”as used herein, in particular refers to nucleic acids that are notcontained in cells. Respective extracellular nucleic acids are alsooften referred to as cell-free nucleic acids. These terms are used assynonyms herein. Hence, extracellular nucleic acids usually are presentexterior of a cell or exterior of a plurality of cells within a sample.The term “extracellular nucleic acids” refers e.g. to extracellular RNAas well as to extracellular DNA. Examples of typical extracellularnucleic acids that are found in the cell-free fraction (respectivelyportion) of biological samples such as e.g. body fluids include but arenot limited to mammalian extracellular nucleic acids such as e.g.extracellular tumor-associated or tumor-derived DNA and/or RNA, otherextracellular disease-related DNA and/or RNA, epigenetically modifiedDNA, fetal DNA and/or RNA, small interfering RNA such as e.g. miRNA andsiRNA, and non-mammalian extracellular nucleic acids such as e.g. viralnucleic acids, pathogen nucleic acids released into the extracellularnucleic acid population e.g. from prokaryotes (e.g. bacteria), viruses,eukaryotic parasites or fungi. The extracellular nucleic acid populationusually comprises certain amounts of intracellular nucleic acids thatwere released from damaged or dying cells. E.g. the extracellularnucleic acid population present in blood usually comprises intracellularglobin mRNA that was released from damaged or dying cells. This is anatural process that occurs in vivo. Such intracellular nucleic acidpresent in the extracellular nucleic acid population can even serve thepurpose of a control in a subsequent nucleic acid detection method. Thestabilization method described herein in particular reduces the riskthat the amount of intracellular nucleic acids, such as genomic DNA,that is comprised in the extracellular nucleic acid population issignificantly increased after the cell-containing sample was collecteddue to the ex vivo handling of the sample. Thus, alterations of theextracellular nucleic acid population because of the ex vivo handlingare reduced and can even be prevented. According to one embodiment, thecell-containing biological sample is or is derived from a body fluidsuch as e.g. blood, plasma, serum, saliva, urine, liquor, cerebrospinalfluid, sputum, lachrymal fluid, sweat, amniotic fluid or lymphaticfluid. Herein, we refer to extracellular nucleic acids that are obtainedfrom a circulating body fluid such as blood or lymphatic fluid ascirculating extracellular nucleic acids or circulating cell-free nucleicacids. According to one embodiment, the term extracellular nucleic acidsin particular refers to mammalian extracellular nucleic acids. Examplesinclude but are not limited to disease-associated or disease-derivedextracellular nucleic acids such e.g. as tumor-associated ortumor-derived extracellular nucleic acids, extracellular nucleic acidsreleased due to inflammations or injuries, in particular traumata,extracellular nucleic acids related to and/or released due to otherdiseases, or extracellular nucleic acids derived from a fetus. The term“extracellular nucleic acids” or “extracellular nucleic acid” asdescribed herein also refers to extracellular nucleic acids obtainedfrom other cell-containing biological samples, in particular biologicalsamples other than body fluids. Usually, a sample comprises more thanone kind or type of extracellular nucleic acids.

The term “extracellular nucleic acid population” as used herein inparticular refers to the collective of different extracellular nucleicacids that are comprised in a cell-containing sample. A cell-containingsample usually comprises a characteristic and thus unique extracellularnucleic acid population. Thus, the type, kind, ratio and/or the theamount of one or more extracellular nucleic acids comprised in theextracellular nucleic acid population of a specific sample may beimportant sample characteristics. As discussed above, it is thereforeimportant to stabilize and thus to substantially preserve saidextracellular nucleic acid population at the state wherein the sample iscollected, as its composition and/or the amount of one or moreextracellular nucleic acids comprised in the extracellular nucleic acidpopulation of a sample can provide valuable medical, prognostic ordiagnostic information. Therefore, it is advantageous if the profile ofthe extracellular nucleic acid population is efficiently stabilized overthe intended stabilization period. The stabilization technologiesdescribed herein reduce contaminations and hence a dilution of theextracellular nucleic acid population by intracellular nucleic acids, inparticular by genomic DNA, after sample collection and stabilization.Thus, a substantial preservation of the extracellular nucleic acidpopulation is achieved. As is shown by the examples, changes in theextracellular nucleic acid population with respect to the quantity, thequality and/or the composition of the comprised extracellular nucleicacids, in particular changes attributable to an increase of releasedgenomic DNA, are over the stabilization period considerably reducedcompared to an unstabilized sample or a corresponding sample that ise.g. stabilized by EDTA in case of a blood sample or a sample derivedfrom blood. According to one embodiment the increase in genomic DNA fromT₀ (stabilization point) to a end of the stabilization period(preferably 48 h, 72 h or 96 h after T₀) is reduced by at least 60%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90% or atleast 95% compared to an unstabilized sample or a corresponding samplethat is e.g. stabilized by EDTA in case of a blood sample (e.g. 1.5 mgEDTA/ml stabilized blood sample) or a sample derived from blood. As isdemonstrated by the examples, values above 80% and higher are achieved,in particular in embodiments, wherein a caspase inhibitor and at leastone primary, secondary or tertiary amide is used in combination.

As is described above and as is demonstrated by the examples, using themethods of the present invention allows for stabilizing thecell-containing sample without refrigeration or freezing for a prolongedtime period. Thus, the samples can be kept at room temperature or evenat elevated temperatures e.g. up to 30° C. or even up to 40° C.According to one embodiment, a stabilization effect is achieved for atleast three days, preferably at least four days, more preferred at least6 days. Preferably, during said stabilization periods the stabilizationmethod according to the invention has the effect that cells contained inthe sample are stabilized, that the release of genomic DNA from cellscontained in the sample into the cell-free portion of the sample isreduced and/or that a degradation of nucleic acids present in the sampleis reduced due to the stabilization. In particular, during the describedstabilization periods, the stabilization reduces the dilution of theextracellular DNA population comprised in the biological sample withgenomic DNA originating from cells contained in the stabilized sampleduring the stabilization period. The stabilizing effects that can beachieved with the method according to the present invention weredescribed in detail above and it is referred to the above disclosure.Preferably, during said stabilization periods, the stabilization reducesthe contamination of the extracellular nucleic acid population comprisedin the biological sample with intracellular nucleic acids, in particulargenomic DNA, originating from cells contained in the stabilized sampleduring the stabilization period. As is shown in the examples, bloodsamples could be stabilized up to 3 days or longer at room temperature.Even during longer storages at room temperature for up to 6 days andeven longer, the extracellular nucleic acid population was substantiallystabilized (in particular compared to non-stabilized samples or e.g.compared to samples that were stabilized using standard methods such asan EDTA treatment) when using a poly(oxyethylene) polymer, preferably incombination with a caspase inhibitor and one or more primary, secondaryor tertiary amides. As is demonstrated by the examples, even over verylong stabilization periods of 10 days the stabilization effect wasmaintained. Generally, it may occur though that the stabilization effectmay decrease over time, which may also depend on the source, e. g. thedonor from which the cell-containing biological sample is derived, itgenerally will be still sufficient to preserve the composition of theextracellular nucleic acid population to allow the analysis and/orfurther processing of the extracellular nucleic acids. Thus,cell-containing biological samples that were stabilized according to themethods of the present invention and in particular samples that werestabilized with a poly(oxyethylene) polymer, one or more primary,secondary or tertiary amides and a caspase inhibitor were still suitablefor isolating and analysing the extracellular nucleic acids containedtherein even after prolonged storage at room temperature. Thus, evenlonger storage/shipping times are conceivable. However, usually, longerperiods are not necessary, as the regular storage and e.g. shipping timeto the laboratory, wherein the nucleic acid isolation and optionally theanalysis is performed, usually does not exceed 6 or 7 days, but usuallyis even completed after two or three days. As is shown in the examples,the stabilization efficiency is particularly good during this timeperiod. However, the long stabilization times and stabilizationefficiencies that are achievable with the method according to thepresent invention provides an important safety factor.

The methods and also the subsequently described stabilizing compositionsaccording to the present invention allow the stabilization of largevolumes of cell-containing biological samples with small volumes/amountsof added stabilizer because the at least one poly(oxyethylene) polymerwhich preferably is a polyethylene glycol and the described combinationsof stabilizers used according to the teachings of the present inventionfor stabilization are highly active in particular in combination. Thisis an important advantage because the size/volume of the sample posesconsiderable restrains on the subsequent nucleic acid isolationprocedure in particular when intending to use automated processes forisolating the extracellular nucleic acids contained in the samples.Furthermore, one has to consider that extracellular nucleic acids areusually only comprised in small amounts in the cell-containingbiological sample. Thus, processing larger volumes of a cell-containingbiological sample such as e.g. a blood sample has the advantage thatmore extracellular nucleic acids can be isolated from the sample andthus are available for a subsequent analysis. According to oneembodiment, the sample volume that is stabilized using the method of thefirst aspect is selected from 1 to 50 ml, 2 to 35 ml, 3 to 25 ml, 4 to20 ml and 5 to 15 ml.

The stabilization methods as disclosed herein, provide a significantadvantage over state-of-the-art stabilization methods that are used forstabilizing the extracellular nucleic acid population in acell-containing sample which are based on the use of cross-linkingreagents, such as formaldehyde, formaline, formaldehyde releasers andthe like. Crosslinking reagents cause inter- or intra-molecular covalentbonds between nucleic acid molecules or between nucleic acids andproteins. This cross-linking effect can significantly impair thesubsequent isolation of nucleic acids from such stabilized samples andusually requires specifically adapted nucleic acid isolation proceduresthat allow the isolation from such samples. As, for example, theconcentration of circulating nucleic acids in a whole blood sample isalready relatively low, any measure which further reduces the yield ofsuch nucleic acids should be avoided. This may be of particularimportance when detecting and analyzing very rare nucleic acid moleculesderived e.g. from malignant tumors or from a developing fetus in thefirst trimester of pregnancy. As is shown by the examples, the method ofthe invention does not require cross-linking agents for stabilization.Therefore, according to one embodiment, the stabilization methodaccording to the present invention does not involve the use of across-linking agent that induces protein-nucleic acid and/orprotein-protein crosslinks. In particular, the stabilization does notinvolve the use of formaldehyde, formaline, paraformaldehyde or aformaldehyde releaser. Furthermore, as described above, according to oneembodiment, the stabilization method according to the invention does notinvolve the use of additives that classify as toxic agents.

After the stabilization period, the method may comprise one or more ofthe following

-   -   a) the stabilized sample is subjected to a nucleic acid analysis        and/or detection method;    -   b) extracellular nucleic acids are isolated from the stabilized        sample;    -   c) extracellular nucleic acids are isolated from the stabilized        sample and the isolated nucleic acids are analysed and/or        detected;    -   d) cells comprised in the stabilized sample are removed;    -   e) cells comprised in the stabilized sample are removed prior to        performing an nucleic acid isolation, analysis and/or detection        step;    -   f) cells are removed from the stabilized sample and        extracellular nucleic acids are isolated from the cell-free or        cell-depleted portion of the stabilized sample;    -   g) (i) the stabilized sample, (ii) the stabilized sample from        which cells have been removed and/or (iii) cells removed from        the sample are stored;    -   h) cells are removed from the stabilized sample and are        discarded; and/or    -   i) cells are removed from the stabilized sample and nucleic        acids are isolated from cells that were removed from the        stabilized sample;    -   j) cells are removed from the stabilized sample and        extracellular nucleic acids are isolated from the cell-free or        cell-depleted portion of the stabilized sample using a size        selective nucleic acid isolation method.

Hence, the cell-containing biological sample that was stabilized usingthe method of the present invention can be analysed in a nucleic acidanalytic and/or detection method and/or may be further processed. Thestabilization of the biological sample may either be followed directlyby techniques for analysing nucleic acids, or nucleic acids may first beisolated from the stabilized sample. Details regarding the nucleic acidisolation and analysis are also described below in conjunction with thesecond aspect of the present invention and it is referred to saiddisclosure.

B. Nucleic Acid Isolation Method

According to a second aspect, a method for isolating extracellularnucleic acids from a cell-containing biological sample is providedcomprising the steps of

-   -   a) stabilizing the cell-containing biological sample according        to the method defined in the first aspect of the present        invention; and    -   b) isolating extracellular nucleic acids from the stabilized        sample.

In step a), the extracellular nucleic acid population comprised in thecell-containing sample is stabilized according to the method describedin the first aspect of the present invention. As discussed above, thestabilization according to the present invention has the effect that theextracellular nucleic acid population contained in the sample may besubstantially preserved in the state it had shown at the time thebiological sample was obtained, respectively collected, over thestabilization period. In particular, the usually observed high increasein nucleic acids that results from intracellular nucleic acids, inparticular genomic DNA, more specifically fragmented genomic DNA, duringstorage/handling is efficiently reduced or even prevented as isdemonstrated in the examples. Without being bound in theory, it isbelieved that the poly(oxyethylene) polymer based stabilizationdescribed herein stabilizes cells and/or reduces the destruction ofcells during the stabilization period, thereby reducing the release ofintracellular nucleic acids. The method allows to separate a cellfraction from the stabilized sample after the desired stabilizationperiod. Hence, extracellular nucleic acids obtained from a respectivelystabilized sample comprise significantly less contamination withintracellular nucleic acids originating from degraded or dying cells andin particular comprise less amounts of fragmented genomic DNA comparedto non-stabilized samples. Furthermore, the stabilization according tothe present invention does not require and preferably does not involvethe use of cross-linking agents. This is an important advantage overprior art methods which involve the use of cross-linking agents such asformaldehyde, formaline or formaldehyde releasers, because thesereagents often reduce the recoverable amount of extracellular nucleicacids due to cross-linking when using standard nucleic acid isolationtechniques. Furthermore, as described above, the stabilization describedherein allows the sample to be stored and/or handled, e.g. transported,—even at room temperature—for a prolonged period of time prior toseparating the cells contained in the sample and/or prior to isolatingnucleic acids comprised therein in step b). With respect to the detailsof the stabilization that is performed in step a), it is referred to theabove disclosure which also applies here. Non-limiting embodiments areagain described briefly in the following.

According to one embodiment, the cell-containing biological sample suchas e.g. a whole blood sample is stabilized in step a) using

-   -   a high molecular weight poly(oxyethylene) polymer having a        molecular weight of at least 1500 as described above, optionally        in combination with a further poly(oxyethylene) polymer having a        molecular weight that is at least 100 lower than the high        molecular weight polymer, such as a low molecular weight        poly(oxyethylene) polymer having a molecular weight of 1000 or        less,    -   at least one caspase inhibitor,    -   one or more primary, secondary and tertiary amides and    -   optionally, further stabilizing agents and/or additives.

Suitable and preferred embodiments of the stabilization method accordingto the present invention that is performed in step a) and the compoundsused for stabilization were described above and it is referred to theabove disclosure which also applies here. Particularly preferred is theadditional use of a low molecular weight poly(oxyethylene) polymer, atleast one caspase inhibitor, and butanamide and/or anN,N-dialkylpropanamide such as preferably N,N-dimethlypropanamide forstabilization. Preferred is the combination with an anticoagulant,preferably a chelating agent such as EDTA, when stabilizing a wholeblood sample. Furthermore, as described above, according to onealternative of the first aspect, the cell-containing biological sampleto be stabilized is contacted with mono-ethylene glycol (1,2-ethanediol)as stabilizing agent. It is referred to the above disclosure.

If the cell-containing biological sample comprises large amounts ofcells as is e.g. the case with whole blood, the cells are separated fromthe remaining sample in order to obtain a cell-free, respectivelycell-reduced or cell-depleted fraction of the stabilized sample fromwhich the extracellular nucleic acids are then isolated in step b).Thus, according to one embodiment, cells are removed from thecell-containing sample between step a) and step b). This intermediatestep may be obsolete if samples are processed which merely compriseminor amounts of residual cells such as e.g. plasma or serum and/orwherein the extracellular nucleic acid of interest is DNA. Due to thestabilization of the invention, the release of genomic DNA during thestabilization period from the contained cells is reduced or evenprevented and furthermore, in particular when using a caspase inhibitorin addition, the fragmentation of genomic DNA is reduced. As describedherein, due to its considerably larger size, unfragmented genomic DNAcan be distinguished from the smaller extracellular DNA. This allows toselectively isolate extracellular DNA even in the presence ofunfragmented genomic DNA by using a size selective isolation protocol.However, in order improve the results, it is preferred that cells (orpotentially remaining cells) are removed from the stabilized sampleprior to isolating the extracellular nucleic acids in step b) in orderto reduce contaminations of the extracellular nucleic acid populationwith intracellular nucleic acids that would otherwise be released fromthe cells during nucleic acid isolation. To remove the contained cellsis also advantageous if the extracellular nucleic acids of interest areRNA, because it can be difficult to distinguish intracellular RNA fromextracellular RNA and furthermore, a dilution of the extracellular RNAcan thereby be prevented. A cell removal step prior to step b) isgenerally advantageous and thus preferred, also if the extracellularnucleic acid of interest is DNA, because this allows to use standardnucleic acid isolation procedures in step b).

Depending on the type of cell-containing biological sample, cells,including residual cells, can be separated and removed e.g. bycentrifugation, preferably high speed centrifugation, or by using meansother than centrifugation, such as e.g. filtration, sedimentation orbinding to surfaces e.g. on (optionally magnetic) particles if acentrifugation step is to be avoided. Respective cell separation methodsare well-known in the prior art and thus, do not need to be described indetail. Respective cell removal steps can also be easily included intoan automated sample preparation protocol. Respectively removed cells mayalso be processed further if desired. The cells can e.g. be stored,analysed and/or biomolecules such as e.g. nucleic acids or proteins canbe isolated from the removed cells. Furthermore, it was found thatintracellular nucleic acids such as intracellular RNA can be stabilizedin particular when additionally using a compound according to formula 1such as DMPA for stabilizing the cell-containing sample. The additionalstabilization of the transcriptome is advantageous as it allows e.g. toanalyse profiles of transcripts in the isolated intracellular nucleicacids which can also be important biomarkers for in vitro diagnostics.

Furthermore, it is also within the scope of the present invention toinclude further intermediate steps to work up the stabilized sample.

Extracellular nucleic acids are isolated in step b), preferably from thecell-free, respectively cell-depleted fraction of the stabilized sample,e.g. from supernatants or from plasma and/or serum in case thestabilized cell-containing sample was a blood sample. For isolatingextracellular nucleic acids, any known nucleic acid isolation method canbe used that is suitable for isolating nucleic acids from the stabilizedsample, respectively the obtained cell-depleted sample. Examples forrespective purification methods include but are not limited toextraction, solid-phase extraction, silica-based purification methods,magnetic particle-based purification, phenol-chloroform extraction,chromatography, anion-exchange chromatography (using anion-exchangesurfaces), electrophoresis, filtration, precipitation and combinationsthereof. It is also within the scope of the present invention tospecifically isolate specific target extracellular nucleic acids, e.g.by using appropriate probes coupled to a solid support that enable asequence specific binding. Also any other nucleic acid isolatingtechnique known by the skilled person can be used.

According to one embodiment, nucleic acids are isolated in step b) usinga chaotropic agent and/or alcohol. Preferably, the nucleic acids areisolated by binding them to a solid phase, preferably a solid phasecomprising silica or carrying anion exchange functional groups.Respective methods are well-known in the prior art and thus, do not needany detailed description. Suitable methods and kits for isolatingextracellular nucleic acids are also commercially available such as theQIAamp® Circulating Nucleic Acid Kit (QIAGEN), the Chemagic CirculatingNA Kit (Chemagen), the NucleoSpin Plasma XS Kit (Macherey-Nagel), thePlasma/Serum Circulating DNA Purification Kit (Norgen Biotek), thePlasma/Serum Circulating RNA Purification Kit (Norgen Biotek), the HighPure Viral Nucleic Acid Large Volume Kit (Roche) and other commerciallyavailable kits suitable for extracting and purifying extracellularnucleic acids. As described above, in particular the embodiment whereina high molecular weight poly(oxyethylene) polymer is used in combinationwith a low molecular weight poly(oxyethylene) polymer is advantageous,as it allows to isolate the extracellular nucleic acids with high yieldusing a broad range of nucleic acid isolation procedures. As describedabove, e.g. in combination with a subsequent nucleic acid isolationmethod that involves the use of a silica column, it is preferred to usea high molecular weight poly(oxyethylene) polymer having a molecularweight of at least 2000, preferably at least 3000, more preferred in arange of 4500 to 10000, in combination with a low molecular weightpoly(oxyethylene) polymer having a molecular weight of 1000 or less,preferably in a range of 150 to 700, more preferred 200 to 600. Asdescribed above, this combination of polymers allows to reduce theamount of high molecular weight poly(oxyethylene) polymer required forefficient stabilization to e.g. 1.5% (w/v) or less, 1.25% (w/v) or less,1% (w/v) or less or also 0.75% (w/v) or less in the stabilized mixturethat contains the cell-containing sample to be stabilized. In the testedexamples, these lower concentrations of the high molecular weightpolymer in combination with the low molecular weight polymersubstantially showed no impairment on the subsequent nucleic acidisolation even when using silica columns, while achieving a strongstabilization effect. Furthermore, as a combination of polymers wasused, also the amount of low molecular weight polymer could be loweredso that the required volume of stabilization composition was kept in anacceptable range. However, as is demonstrated in the examples,respective lower concentrations of the high molecular weightpoly(oxyethylene) polymer of 1.5% (w/v) or less, 1.25% (w/v) or less, 1%(w/v) or less or also 0.75% (w/v) or less may also be used in theabsence of a low molecular weight poly(oxyethylene) polymer, if furtherstabilizing agents are used, preferred embodiments of such stabilizingagents are described herein.

According to one embodiment, nucleic acids are isolated in step b) bybinding them to a solid phase comprising anion exchange groups. Suitableanion exchange groups are for example provided by amine groups. Bindingpreferably occurs at a pH below 7. Such anion exchange based nucleicacid isolation methods are known to the skilled person. According to oneembodiment, the nucleic acids are extracellular nucleic acids. Suitableanion exchange based methods are e.g. described in WO 2013/045432,herein incorporated by reference. The described method is particularlysuitable for isolating exctracellular nucleic acids, such asextracellular DNA, from plasma that was obtained from a blood samplethat was stabilized using the stabilization method described herein.

According to one embodiment, total nucleic acids are isolated from thestabilized cell-containing sample that is obtained after step a) oroptionally the sample that is obtained after cells were removed from thestabilized cell-containing sample in an intermediate step. Preferably,the nucleic acids are isolated from the stabilized sample, or acell-free, respectively cell-depleted fraction of the stabilized sample.E.g. total nucleic acids can be isolated from plasma or serum and theextracellular nucleic acids will be comprised as portion in theseextracted nucleic acids. If the cells contained in the stabilized sampleare efficiently removed prior to nucleic acid isolation, the isolatedtotal nucleic acids will predominantly comprise or even consist ofextracellular nucleic acids.

It is also within the scope of the present invention to isolate at leastpredominantly a specific target nucleic acid. A target nucleic acid canbe e.g. a certain type of extracellular nucleic acid, e.g. DNA or RNA,including mRNA, microRNA, other non-coding nucleic acids, epigeneticallymodified nucleic acids, and other nucleic acids. E.g. the targetextracellular nucleic acid can be DNA and the non-target extracellularnucleic acid can be RNA or vice versa. Target specific nucleic acidisolation methods which specifically aim at isolating DNA or RNA arealso well known in the prior art and thus, do not need any detaileddescription herein. According to one embodiment, the non-target nucleicacid is destroyed by adding an appropriate enzyme which specificallydestroys the non-target nucleic acid, e.g. a RNase if the target nucleicacid is DNA or a DNase if the target nucleic acid is RNA. Said enzymecan be added to the lysis or binding mixture or can be added afterextracellular nucleic acids were bound to a solid phase. Suitableembodiments for performing a respective non-target nucleic aciddigestion step are known in the prior art and thus, do not need anyfurther description herein. According to one embodiment which isfeasible if DNA and RNA are bound to a solid support, elution conditionsselective for the target nucleic acid can be applied to predominantlyand thus selectively recover the target nucleic acid from the solidsupport. According to one embodiment, an isolation method is used,wherein the target nucleic acid, e.g. DNA, is selectively bound to asolid phase under conditions wherein non-target nucleic acids, e.g. RNAdo not bind. Suitable binding conditions are well-known in the prior artand are e.g. described in WO 95/21849. According to one embodiment, thenon-target nucleic acid is removed by binding at least a portion of thenon-target nucleic acid under appropriate conditions to a solid phaseand then separating the non-target nucleic acid bound to the solid phasefrom the remaining sample comprising the target extracellular nucleicacid. This can be achieved e.g. by the addition of a suitable solidphase under conditions wherein mainly the non-target nucleic acids e. g.DNA are bound to the solid phase while the non-target nucleic acid, e.g.RNA, remains in the sample and is recovered therefrom in a separatestep. Suitable methods for selectively removing a non-target nucleicacid from a target nucleic acid are for example described in EP 0 880537 and WO 95/21849, herein incorporated by reference. If desired, saidnon-target nucleic acid may also be further used, e.g. further processedsuch as e.g. eluted from the solid phase. However, it may also bediscarded. It is also within the scope of the present invention to e.g.digest the non-target nucleic acid or remainders thereof using nucleasesafter isolation of the target nucleic acid.

The term target nucleic acid may also refer to a specific kind ofnucleic acid, e.g. a specific extracellular nucleic acid that is knownto be a certain disease marker. As discussed above, the isolation ofextracellular nucleic acids may also comprise the specific isolation ofa respective target nucleic acid e.g. by using appropriate captureprobes which support the selective isolation of the target nucleic acid.

The term target nucleic acid may also refer to nucleic acids having acertain length, e.g. a nucleic acid having a length of 5000 nt or less,2000 nt or less, 1000 nt or less, 900 nt or less, 800 nt or less, 700 ntor less, 600 nt or less, 500 nt or less, 400 nt or less or 350 nt orless. Isolating target nucleic acids of a certain maximum size can beadvantageous in the context of the present invention. It is known thatextracellular nucleic acids usually have a size of 1000 nt or less andusually even 500 nt or less. The sizes, respectively size rangesindicated herein refer to the chain length. I.e. in case ofdouble-stranded nucleic acids such as double-stranded DNA it refers tobp. Selectively isolating smaller nucleic acids in step b) can increasethe portion of extracellular nucleic acids obtained in the isolatednucleic acids. The stabilization methods according to the presentinvention allow, in particular due to the inhibition of the release ofgenomic DNA and/or the inhibition of the fragmentation of releasedgenomic DNA, for a more efficient separation of such high molecularweight genomic DNA from the smaller extracellular nucleic acidpopulation. As the substantial size difference between genomic DNA andextracellular nucleic acids is essentially preserved using thestabilization technology according to the present invention, genomic DNAcan be removed more efficiently e.g. using a size selective nucleic acidisolation protocol. As the size difference between genomic DNA (usuallylarger than >10,000 bp) and extracellular nucleic acids (usually <1000nt/bp) in a sample stabilized as described herein is usually relativelylarge due to the efficient stabilization, known methods for selectivelyisolating nucleic acids of a certain target length can be used. Thus,according to one embodiment, step b) comprises selectively isolatingtarget nucleic acids having a length of 2000 nt or less, 1500 nt orless, 1000 nt or less, 900 nt or less, 800 nt or less, 700 nt or less,600 nt or less or 500 nt or less. Suitable methods to achieve arespective size selective isolation of nucleic acids e.g. by depletinghigh molecular weight genomic DNA, are known in the prior art and thus,need no detailed description herein. A classic method for isolating DNAof a target size involves the separation of the DNA in a gel, cuttingout the desired gel band(s) and then isolating the DNA of the targetsize from the gel fragment(s). Another widely used technology is thesize selective precipitation with polyethylene glycol based buffers (Lisand Schleif Nucleic Acids Res. 1975 March; 2(3):383-9) or thebinding/precipitation on carboxyl-functionalized beads (DeAngelis et al,Nuc. Acid. Res. 1995, Vol 23(22), 4742-3; U.S. Pat. No. 5,898,071 andU.S. Pat. No. 5,705,628, commercialized by Beckman-Coulter (AmPure XP;SPRIselect) and U.S. Pat. No. 6,534,262). Furthermore, size selectiveisolation methods that are based on the use of solid supports comprisinganion exchange groups and varying pH values are known. A size selectiveisolation provides further opportunities in order to reduce the amountof intracellular nucleic acids in the isolated extracellular nucleicacids. For example, when the target extracellular nucleic acid ofinterest is DNA, the removal of genomic DNA during nucleic acidisolation step b) could also supplement or even replace a separate highg-force centrifugation of a plasma sample before starting the nucleicacid extraction in order to remove residual cells. Genomic DNA that isreleased from said residual cells is prevented from becoming massivelydegraded due to the stabilization according to the present invention, inparticular if a caspase inhibitor is additionally used, and accordingly,said unfragmented or less fragmented genomic DNA can be depleted byusing a size-selective nucleic acid isolation protocol in step b). Thisoption is of particular advantage, as many clinical laboratories do nothave a centrifuge capable of performing such a high g-forcecentrifugation or other means for removing in particular trace amountsof residual cells.

The isolated extracellular nucleic acids can then be analysed and/orfurther processed in a step c) using suitable assay and/or analyticalmethods. E.g. they can be identified, modified, contacted with at leastone enzyme, amplified, reverse transcribed, cloned, sequenced, contactedwith a probe, be detected (their presence or absence) and/or can bequantified. Respective methods are well-known in the prior art and arecommonly applied in the medical, diagnostic and/or prognostic field inorder to analyse extracellular nucleic acids (see also the detaileddescription in the background of the present invention). Thus, afterextracellular nucleic acids were isolated in step b), optionally as partof total nucleic acids, total RNA and/or total DNA (see above), they canbe analysed e.g. to identify the presence, absence or severity of adisease state including but not being limited to a multitude ofneoplastic diseases, in particular premalignancies and malignancies suchas different forms of tumors or cancers. E.g. the isolated extracellularnucleic acids can be analysed in order to detect diagnostic and/orprognostic markers (e.g., fetal- or tumor-derived extracellular nucleicacids) in many fields of application, including but not limited tonon-invasive prenatal genetic testing respectively screening, diseasescreening, pathogen screening, oncology, cancer screening, early stagecancer screening, cancer therapy monitoring, genetic testing(genotyping), infectious disease testing, injury diagnostics, traumadiagnostics, transplantation medicine or many other diseases and, hence,are of diagnostic and/or prognostic relevance. According to oneembodiment, the isolated extracellular nucleic acids are analyzed toidentify and/or characterize a disease or a fetal characteristic. Thus,as discussed above, the isolation method described herein may furthercomprise a step c) of nucleic acid analysis and/or processing.

Therefore, according to one embodiment, the isolated extracellularnucleic acids are analysed in a step c) to identify, detect, screen for,monitor or exclude a disease and/or at least one fetal characteristic.The analysis/further processing of the isolated extracellular nucleicacids can be performed using any nucleic acid analysis/processing methodincluding, but not limited to amplification technologies, polymerasechain reaction (PCR), isothermal amplification, reverse transcriptionpolymerase chain reaction (RT-PCR), quantitative real time polymerasechain reaction (Q-PCR), digital PCR, gel electrophoresis, capillaryelectrophoresis, mass spectrometry, fluorescence detection, ultravioletspectrometry, hybridization assays, DNA or RNA sequencing, nextgeneration sequencing, restriction analysis, reverse transcription,nucleic acid sequence based amplification (NASBA), allele specificpolymerase chain reaction, polymerase cycling assembly (PCA), asymmetricpolymerase chain reaction, linear after the exponential polymerase chainreaction (LATE-PCR), helicase-dependent amplification (HDA), hot-startpolymerase chain reaction, intersequence-specific polymerase chainreaction (ISSR), inverse polymerase chain reaction, ligation mediatedpolymerase chain reaction, methylation specific polymerase chainreaction (MSP), multiplex polymerase chain reaction, nested polymerasechain reaction, solid phase polymerase chain reaction, or anycombination thereof. Respective technologies are well-known to theskilled person and thus, do not need further description here.

According to one embodiment, either or both of isolation step b) andanalysis step c) occur at least one day up to 3 days or two days up to10 days after the cell-containing biological sample has been collected,respectively was stabilized according to the teachings of the presentinvention. Suitable time periods for which the cell-containingbiological sample, in particular a blood sample, respectively theextracellular nucleic acid population contained therein can bestabilized using the method according to the present invention are alsodescribed above in conjuncton with the stabilization method and therespective disclosure also applies here. According to one embodiment,nucleic acid isolation step b) is performed at least one day, at least 2days or at least 3 days after the cell-containing sample was collectedand stabilized according to the method according to the presentinvention.

C. Stabilization Composition

According to a third aspect, a composition suitable for stabilizing acell-containing biological sample is provided comprising

-   -   i) a poly(oxyethylene) polymer as stabilizing agent or    -   ii) mono-ethylene glycol as stabilizing agent        and one or more, preferably two or more, further additives        selected from the group consisting of    -   one or more primary, secondary or tertiary amides;    -   a caspase inhibitor;    -   an anticoagulant and/or a chelating agent.

Preferably, the composition according to the third aspect comprises apoly(oxyethylene) polymer, which preferably is a high molecular weightpoly(oxyethylene) polymer having a molecular weight of at least 1500, asstabilizing agent and furthermore comprises one or more, preferably twoor more further additives selected from the group consisting of

-   -   at least one further poly(oxyethylene) polymer having a        molecular weight that is at least 100, preferably at least 200,        at least 300 or at least 400 below the molecular weight of the        first poly(oxyethylene) polymer which preferably is a high        molecular weight poly(oxyethylene) polymer, wherein said further        poly(oxyethylene) polymer preferably is a low molecular weight        poly(oxyethylene) polymer having a molecular weight of 1000 or        less;    -   one or more primary, secondary or tertiary amides;    -   a caspase inhibitor;    -   an anticoagulant and/or a chelating agent.

The advantages and suitable and preferred embodiments are discussedabove in conjunction with the stabilization method according to thefirst aspect and it is referred to the above disclosure which alsoapplies here. As discussed above, the stabilizing compositions providedby the invention, in particular those comprising a poly(oxyethylene)polymer, which preferably is a high molecular weight poly(oxyethylene)polymer, and at least one caspase inhibitor, optionally but preferablyin combination with one or more primary, secondary or tertiary amides,are particularly effective in stabilizing a cell-containing biologicalsample, in particular blood, plasma and/or serum, by stabilizingcomprised cells and the comprised extracellular nucleic acids therebysubstantially preserving, respectively stabilizing the extracellularnucleic acid population at the time of stabilization. A respectivestabilizing composition allows the storage and/or handling, e.g.shipping, of the cell-containing biological sample, which preferably isblood, at room temperature for at least two, preferably at least threedays or even longer without substantially compromising the quality ofthe blood sample, respectively the extracellular nucleic acid populationcontained therein. In particular, dilutions, respectively contaminationsof the extracellular nucleic acid composition with intracellular nucleicacids, in particular fragmented genomic DNA, are reduced or evenprevented over the stabilization period. Preferably, the stabilizationcomposition is contacted with the cell-containing sample immediatelyafter or during collection of the cell-containing biological sample.Furthermore, as described above, mono-ethylene glycol can be used eitheralternatively or in addition to the poly(oxyethylene) polymer asstabilizing agent. The preferably used combinations with the otherstabilizing agents are essentially the same.

The stabilization composition comprises according to one embodiment ahigh molecular weight poly(oxyethylene) polymer. Details are describedabove in conjunction with the stabilization method according to thefirst aspect and it is referred to the respective disclosure. The highmolecular weight poly(oxyethylene) polymer is preferably a polyethyleneglycol, such as a unsubstituted polyethylene glycol. In embodiments, themolecular weight may lie in a range selected from 1500 to 40000, 2000 to30000, 2500 to 25000, 3000 to 20000, 3500 to 15000, 4000 to 10000, 4500to 9000 and 5000 to 8000. As described above, also higher molecularweights exceeding 40000 can be used.

According to one embodiment, the stabilization composition comprises apoly(oxyethylene) polymer that has a molecular weight of 1500 or lessand in embodiments is a low molecular poly(oxyethylene) polymer having amolecular weight of 1000 or less. The low molecular weightpoly(oxyethylene) polymer may have a molecular weight that lies in arange selected from 100 to 1000, 150 to 800, 150 to 700, preferably 200to 600 and more preferably 200 to 500 such as 200 to 400. As isdemonstrated by the examples, a stabilization composition comprisingsuch a poly(oxyethylene) polymer, which preferably is a polyethyleneglycol, are effective stabilizers when being used in combination withone or more further stabilizing agents, such as preferably a caspaseinhibitor and one or more primary, secondary or tertiary amides asdescribed herein. The stabilization effect of these stabilizing agentsis improved if the stabilization composition comprises a respectivepoly(oxyethylene) polymer having a molecular weight of 1500 or less,such as a low molecular weight poly(oxyethylene) polymer having amolecular weight of 1000 or less.

According to one embodiment, the stabilization composition comprises ahigh molecular weight poly(oxyethylene) polymer and additionallycomprises at least one further poly(oxyethylene) polymer having amolecular weight that is at least 100, preferably at least 200, at least300 or at least 400 below the molecular weight of the high molecularweight poly(oxyethylene) polymer. According to one embodiment, thedifference in the molecular weight is at least 2500, at least 3500, atleast 5000 or at least 7500. As described above in conjunction with thestabilization method according to the first aspect, using a combinationof poly(oxyethylene) polymers that differ in their molecular weights isadvantageous. Preferably, both poly(oxyethylene) polymers arepolyethylene glycols such as unsubstituted polyethylene glycol.According to an advantageous embodiment, the stabilization compositioncomprising a high molecular weight poly(oxyethylene) polymer having amolecular weight of at least 1500 additionally comprises a low molecularweight poly(oxyethylene) polymer having a molecular weight of 1000 orless. Details are described above in conjunction with the stabilizationmethod according to the first aspect and it is referred to therespective disclosure which also applies here. The low molecular weightpoly(oxyethylene) polymer is preferably a polyethylene glycol, such as aunsubstituted polyethylene glycol. The molecular weight of the lowmolecular weight poly(oxyethylene) polymer may lie in a range selectedfrom 100 to 1000, 150 to 800 and preferably lies in the range of 200 to600.

According to one embodiment, the stabilization composition comprising apoly(oxyethylene) polymer, which preferably is a high molecular weightpoly(oxyethylene) polymer having a molecular weight of at least 1500,additionally comprises one or more primary, secondary or tertiaryamides. The advantages of additionally using one or more of such amidesin combination with a poly(oxyethylene) polymer and suitable andpreferred embodiments were described in detail above in conjunction withthe stabilization method according to the first aspect and it isreferred to the above disclosure which also applies here. According toone embodiment, the at least one primary, secondary or tertiary amidecomprised in the stabilization composition is a compound according toformula 1

wherein R1 is a hydrogen residue or an alkyl residue, preferably a C1-C5alkyl residue, a 01-C4 alkyl residue or a C1-C3 alkyl residue, morepreferred a C1-C2 alkyl residue, R2 and R3 are identical or differentand are selected from a hydrogen residue and a hydrocarbon residue,preferably an alkyl residue, with a length of the carbon chain of 1-20atoms arranged in a linear or branched manner, and R4 is an oxygen,sulphur or selenium residue. Preferably, the amide is a carboxylic acidamide so that R4 is oxygen. Preferred embodiments were described abovein conjunction with the stabilization method and it is referred to theabove disclosure which also applies here. Preferably, a compoundaccording to formula 1 is used which is not classified as toxic agent.Preferably, said stabilization composition comprising apoly(oxyethylene) polymer, which preferably is a high molecular weightpoly(oxyethylene) polymer and comprising a compound according to formula1 additionally comprises a caspase inhibitor.

The compound according to formula 1 may be a carboxylic acid amideselected from primary carboxylic acid amides and secondary carboxylicacid amides. According to one embodiment, the composition comprises aprimary carboxylic acid amide selected from the group consisting offormamide, acetamide, propanamide and butanamide. Preferably, thecarboxylic acid is selected from butanamide and formamide. Morepreferred, it is butanamide, as this agent is particularly effective forstabilizing the extracellular nucleic acid population.

According to one embodiment, the at least one compound according toformula 1 is a N,N-dialkyl-carboxylic acid amide. According to oneembodiment, the compound according to formula 1 is aN,N-dialkylpropanamide, preferably N,N-dimethlypropanamide.N,N-dimethylpropanamide is not classified as toxic agent. Furthermore,N,N-dimethylpropanamide has the advantageous effect that it isadditionally capable of stabilizing intracellular nucleic acids, and inparticular may stabilize transcript profiles if used in an appropriateconcentration.

According to one embodiment, the stabilization composition comprisesbutanamide and/or an N,N-dialkylpropanamide, wherein saidN,N-dialkylpropanamide preferably is N,N-dimethylpropanamide. As isdemonstrated by the examples, both amides alone and in combinationsignificantly improve the observed stabilization effect.

According to one embodiment, the stabilization composition comprising apoly(oxyethylene) polymer additionally comprises a caspase inhibitor.The advantages of using a caspase inhibitor in combination and suitableand preferred embodiments of the caspase inhibitor were described indetail above in conjunction with the stabilization method according tothe first aspect and it is referred to the above disclosure which alsoapplies here. Preferably, the caspase inhibitor is a pancaspaseinhibitor. Preferably, the caspase inhibitor is a modified caspasespecific peptide, preferably modified at the C-terminus with anO-phenoxy group such as Q-VD-OPh. According to an advantageousembodiment, a high molecular weight poly(oxyethylene) polymer having amolecular weight of at least 1500 is used as poly(oxyethylene) polymer.Preferably, polyethylene glycol is used. Preferably, unsubstitutedpolyethylene glycol is used.

According to one embodiment, the stabilization composition comprises apoly(oxyethylene) polymer, which preferably is a high molecular weightpoly(oxyethylene) polymer, and at least one anticoagulant. Thisembodiment is particularly suitable for stabilizing a blood sample or acell-containing sample derived from blood. According to one embodiment,the stabilization composition comprises a poly(oxyethylene) polymer,which preferably is a high molecular weight poly(oxyethylene) polymer,and a chelating agent. Suitable chelating agents which also function asanticoagulant as well as suitable concentrations for stabilization weredescribed above in conjunction with the method according to the presentinvention and it is referred to the above disclosure which also applieshere. Preferably, EDTA is used as chelating agent.

The stabilization composition may also comprise further additives asdescribed above in conjunction with the stabilization method.

For stabilizing blood, the stabilization composition preferablycomprises a poly(oxyethylene) polymer, which preferably is a highmolecular weight poly(oxyethylene) polymer, at least one caspaseinhibitor and an anticoagulant and optionally at least one primary,secondary or tertiary amide as described above. The poly(oxyethylene)polymer is preferably polyethylene glycol. As described, said amide ispreferably a compound according to formula 1. The use of butanamideand/or a N,N-dialkylpropanamide, preferably N,N-dimethlypropanamide ispreferred. According to one embodiment, said composition comprises ahigh molecular weight poly(oxyethylene) polymer, which preferably is apolyethylene glycol, and additionally comprises a low molecular weightpoly(oxyethylene) polymer, which preferably is a polyethylene glycol.Preferred molecular weights were described above and it is referred tothe respective disclosure.

According to one embodiment, the stabilization composition comprises

-   -   a) at least one high molecular weight poly(oxyethylene) polymer        having a molecular weight of at least 1500, preferably in a        range of 1500 to 50000, 2000 to 40000, 2500 to 30000, 2500 to        25000, more preferred 3000 to 20000, 3500 to 15000 or 4500 to        10000;    -   b) one or more compounds according to formula 1;    -   c) at least one caspase inhibitor, preferably a pancaspase        inhibitor, more preferred Q-VD-OPh    -   d) at least one further poly(oxyethylene) polymer having a        molecular weight that is at least 100, preferably at least 200,        at least 300 or at least 400 below the molecular weight of the        high molecular weight poly(oxyethylene) polymer used and wherein        said further poly(oxyethylene) polymer preferably is a low        molecular weight poly(oxyethylene) having a molecular weight of        1000 or less, preferably having a molecular weight in a range of        200 to 800 or 200 to 600;    -   e) optionally an anticoagulant and/or a chelating agent, more        preferably EDTA.

According to one embodiment, the stabilization composition comprises

-   -   a) at least one high molecular weight poly(oxyethylene) polymer        having a molecular weight that lies in the range of 2000 to        40000, 2500 to 30000, 2500 to 25000, 3000 to 20000 or 3500 to        15000;    -   b) one or more compounds according to formula 1, preferably        butanamide and/or N,N-dimethlypropanamide;    -   c) at least one caspase inhibitor;    -   d) at least one low molecular weight poly(oxyethylene) polymer        having a molecular weight that lies in a range of 100 to 1000,        150 to 800 or 200 to 600;    -   e) an anticoagulant and/or a chelating agent, preferably EDTA.

According to one embodiment, the stabilization composition comprises

-   -   a) at least one high molecular weight poly(oxyethylene) polymer        having a molecular weight that lies in the range of 4500 to        10000;    -   b) one or more compounds according to formula 1, preferably        butanamide and/or N,N-dimethlypropanamide;    -   c) at least one caspase inhibitor;    -   d) at least one low molecular weight poly(oxyethylene) polymer        having a molecular weight that lies in a range of 100 to 800,        preferably 200 to 600;    -   e) an anticoagulant and/or a chelating agent, preferably EDTA.

Suitable and preferred embodiments of the high and low molecular weightpoly(oxyethylene) polymer, the caspase inhibitor, the compound accordingto formula 1 and anticoagulants as well as chelating agents weredescribed in detail above in conjunction with the stabilization methodand it is referred to the above disclosure which also applies here.Preferably, the anticoagulant is a chelating agent, more preferablyEDTA.

Suitable and preferred concentrations of the individual agents that canbe used for stabilization in the stabilization mixture comprising thestabilizing composition and the cell-containing biological sample weredescribed above and also apply here. The skilled person can choseappropriate concentrations of said agents in the stabilizationcomposition to achieve said concentrations in the mixture comprising thecell-containing sample when the intended amount of the stabilizationcomposition is mixed with the intended amount of cell-containing sampleto be stabilized. It is referred to the above disclosure which alsoapplies here with respect to the stabilization composition.

According to one embodiment, the stabilization composition is a liquid.Subsequently, concentrations of the individual agents are indicated, ifpresent in the stabilization composition, that are particularlypreferred for the stabilisation of blood samples. E.g. a liquidstabilisation composition of 0.5 ml to 2.5 ml, 0.5 ml to 2 ml,preferably 1 ml to 2 ml or 1 ml to 1.5 ml can be used. Suchstabilization composition comprising the stabilizing agents in theconcentrations indicated below, can be used for stabilizing e.g. 10 mlblood.

According to one embodiment, said liquid stabilization compositioncomprises a high molecular weight poly(oxyethylene) polymer whichpreferably is a polyethylene glycol in a concentration selected from0.4% to 35% (w/v), 0.8% to 25% (w/v), 1.5% to 20% (w/v), 2.5% to 17.5%(w/v), 3% to 15% (w/v), 4% to 10% (w/v) and 3% to 5% (w/v). Suitableconcentrations can be determined by the skilled person and may interalia depend on whether the high molecular weight poly(oxyethylene)glycol is used as alone or in combination with a furtherpoly(oxyethylene) polymer such as a low poly(oxyethylene) polymer andthe amount, e.g. the volume, of the stabilization composition used tostabilize a certain amount of cell-containing sample. Examples ofconcentration ranges suitable when using a high molecular weightpoly(oxyethylene) polymer alone include but are not limited toconcentrations selected from 2.2% to 33.0% (w/v), 4.4% to 22.0 (w/v) %,6.6% to 16.5% (w/v) and 8.8% to 13.2% (w/v). Examples of concentrationranges suitable when using a high molecular weight poly(oxyethylene)polymer in combination with a low molecular weight poly(oxyethylene)polymer include but are not limited to concentrations selected from 0.4%to 30.7%, 0.8% to 15.3%, 1% to 10%, 1.5% to 7.7%, 2.5% to 6%, 3.1% to5.4% and 3% to 4%.

In a specific embodiment, the liquid stabilization composition comprises5 mg to 500 mg, in particular 10 mg to 250 mg, 25 mg to 150 mg, or 40 mgto 100 mg of the high molecular weight poly(oxyethylene) polymer whichpreferably is a polyethylene glycol. In particular, the liquidstabilization composition may comprise 0.5 μmol to 50 μmol, inparticular 1 μmol to 25 μmol, 2 μmol to 20 μmol, or 3 μmol to 10 μmol ofthe high molecular weight poly(oxyethylene) polymer. Such liquidstabilization composition can be filled e.g. in a collection device andis e.g. suitable for stabilizing a sample unit.

According to one embodiment, said liquid stabilization compositioncomprises a low molecular weight poly(oxyethylene) polymer, whichpreferably is a polyethylene glycol in a concentration selected from0.8% to 92.0%, 3.8% to 76.7%, 11.5% to 53.7%, 19.2% to 38.3%, 20% to 30%and 20% to 27.5%. The aforementioned concentrations refer to (w/v) or(v/v) depending on whether the low molecular weight poly(oxyethylene)polymer is a liquid or not. As is demonstrated in the examples, lowmolecular weight poly(oxyethylene) polymers can efficiently support thestabilization of cell-containing samples, in particular when being usedin combination with one or more further stabilizing agents as describedherein.

In a specific embodiment, the liquid stabilization composition comprises40 μl to 4000 μl, in particular 100 μl to 2000 μl, 150 μl to 1500 μl,200 μl to 1000 μl or 250 μl to 750 μl of the low molecular weightpoly(oxyethylene) polymer. In particular, the liquid stabilizationcomposition may comprise 0.2 mmol to 15 mmol, in particular 0.5 mmol to10 mmol, 0.75 mmol to 5 mmol, 1 mmol to 3 mmol, or 1.2 mmol to 2 mmol ofthe low molecular weight poly(oxyethylene) polymer. Such liquidstabilization composition can be filled e.g. in a collection device andis e.g. suitable for stabilizing a sample unit.

According to one embodiment, said liquid stabilization compositioncomprises one or more primary, secondary or tertiary amides in aconcentration selected from 0.4% to 38.3%, 0.8% to 23.0%, 2.3% to 11.5%,3.8% to 9.2%, 5% to 15% and 7.5% to 12.5%. The aforementionedconcentrations refer to (w/v) or (v/v) depending on whether the primary,secondary or tertiary amide is a liquid or not. As described above, itis preferred that the stabilizing composition additionally comprises oneor more primary, secondary or tertiary amides and suitable and preferredexamples were described above.

In a specific embodiment, the liquid stabilization composition comprises10 μl to 2000 μl, in particular 50 μl to 1000 μl, 100 μl to 750 μl, or125 μl to 500 or 150 to 250 μl of the primary, secondary or tertiaryamide. In particular, the liquid stabilization composition may comprise0.2 mmol to 15 mmol, in particular 0.5 mmol to 10 mmol, 0.75 mmol to 5mmol, 1 mmol to 3 mmol, or 1.2 mmol to 2 mmol of the primary, secondaryor tertiary amide. Such liquid stabilization composition can be fillede.g. in a collection device and is e.g. suitable for stabilizing asample unit.

According to one embodiment, said liquid stabilization compositioncomprises a caspase inhibitor in a concentration selected from 0.1 μM to220 μM, 0.8 μM to 115.0 μM, 7.7 μM to 76.7 μM and 23.0 μM to 50 μM. In aspecific embodiment, the liquid stabilization composition comprises 1nmol to 1000 nmol, in particular 5 nmol to 500 nmol, 10 nmol to 200 nmolor 25 nmol to 100 nmol of the caspase inhibitor. Such liquidstabilization composition can be filled e.g. in a collection device andis e.g. suitable for stabilizing a sample unit.

According to one embodiment, said liquid composition comprises achelating agent, preferably EDTA such as K₂EDTA in a concentrationselected from 9.5 mM to 1100 mM, 20 mM to 750 mM, 50 mM to 600 mM, 75 mMto 550 mM, 100 mM to 500 mM, 125 mM to 450 mM, 130 mM to 300 mM and 140mM to 250 mM. In a specific embodiment, the liquid stabilizationcomposition comprises 10 nmol to 3000 nmol, in particular 50 nmol to1500 nmol, 100 nmol to 1000 nmol or 150 nmol to 500 nmol of thechelating agent. Such liquid stabilization composition can be fillede.g. in a collection device and is e.g. suitable for stabilizing asample unit.

As described above, said liquid stabilization composition comprises apoly(oxyethylene) polymer which preferably is a high molecular weightpoly(oxyethylene) polymer, and preferably one or more primary, secondaryor tertiary amides, a caspase inhibitor and the chelating agent.According to one embodiment, said liquid composition comprises a highmolecular weight poly(oxyethylene) polymer and comprises additionally alow molecular weight poly(oxyethylene) glycol.

According to one alternative, the composition comprises mono-ethyleneglycol (1,2-ethanediol) as stabilizing agent. Mono-ethylene glycol maybe used in the concentrations described above for the low molecularweight poly(oxyethylene) polymer. It is referred to the repectivedisclosure which also applies here. In particular, the composition maycomprise mono-ethylene glycol and any one or more of the otherstabilizing agents described above in conjunction with the composition.According to one embodiment, the composition a comprises additionally atleast one caspase inhibitor and/or at least one primary, secondary ortertiary amide, in particular a compound according to formula 1 asdescribed above. Preferably, the composition comprises both. Thecomposition comprising mono-ethylene glycol may also comprise ananticoagulant, preferably a chelating agent, and/or a poly(oxyethylene)polymer, such as a high and/or low molecular weight poly(oxyethylene)polymer, as described above. Suitable embodiments and concentrationranges for the respective stabilizing agents are described above andalso apply to the embodiment, wherein the composition comprisesmono-ethylene glycol as stabilizing agent.

The stabilizing composition provided by the present invention stabilizesthe cell-containing biological sample and thus, does not induce thelysis and/or disruption of nucleated cells and preferably also anucleated cells, contained in the sample. Therefore, the stabilizationcomposition does not comprise additives in a concentration wherein saidadditives would induce or promote cell lysis of respective cells andpreferably cells in general. The stabilizing composition reduces thedamage of cells comprised in the sample as can be e.g. determined by theassay methods described in the example section. In particular, thestabilization composition described herein is capable of reducing therelease of genomic DNA from cells contained in the cell-containingbiological sample into the cell-free portion of the sample. Furthermore,in particular when additionally comprising a caspase inhibitor what ispreferred, the stabilization composition may be capable of reducing thedegradation of nucleic acids, in particular genomic DNA, present in thestabilized sample. As described, the stabilization composition iscapable of reducing or preventing the contamination of the extracellularDNA population comprised in the biological sample with genomic DNAoriginating from cells contained in the stabilized sample. Preferably,it is capable of reducing or preventing the contamination of theextracellular nucleic acid population comprised in the biological samplewith intracellular nucleic acids, in particular DNA and RNA, originatingfrom cells contained in the stabilized sample. Preferably, thestabilization composition does not comprise a cross-linking agent thatinduces protein-DNA and/or protein-protein crosslinks. In particular,the stabilization composition does not comprise formaldehyde, formaline,paraformaldehyde or a formaldehyde releaser or similar crosslinkingagents. Preferably, the stabilization composition does not compriseagents that are classified as toxic agents according to GHS.

Preferably, the stabilization composition of the invention is capable ofstabilizing the extracellular nucleic acid population comprised in thecell-containing biological sample without refrigeration, preferably atroom temperature, for a time period selected from at least two days, atleast three days, at least four days, at least five days and/or at leastsix days. In particular, one or more, preferably all of theabove-described stabilizing effects are achieved during the definedstabilization periods.

According to one embodiment, the stabilizing composition is for thestabilization of blood and consists essentially of the one or morepoly(oxyethylene) polymers, one or more primary, secondary or tertiaryamides, the at least one caspase inhibitor, and an anticoagulant, whichpreferably is a chelating agent such as EDTA and optionally, a solventand/or buffering agent. As described, preferably water is used assolvent as it reduces hemolysis during the storage period. The sameapplies mutatis mutandis to the embodiment wherein mono-ethylene glycolis used as stabilizing agent.

The stabilization composition may be provided in a solid form, asemi-liquid form or as liquid. A solid composition is e.g. a suitableoption if the cell-containing biological sample to be stabilizedcontains liquid to dissolve the solid (such as for examplecell-containing body fluids, cells in medium, urine) or if liquid, e.g.water is added thereto to dissolve the solid composition. As isdemonstrated by the examples, the stabilising composition can be used insolid-form. The advantage of using a solid stabilizing composition isthat solids are usually chemically more stable. However, also a liquidstabilization composition may be used. Liquid compositions have theadvantage that the mixture with the sample to be stabilised can bequickly achieved, thereby basically providing an immediate stabilizingeffect as soon as the sample comes into contact with the liquidstabilizing composition. Preferably, stabilizing agent(s) present in theliquid stabilizing composition remain stable in solution and require nopre-treatment-such as for example the dissolving of precipitates oflimited solubility-by the user because pre-treatments of this kind posea risk of variations in the stabilizing efficiency. As is demonstratedby the examples, a stabilization composition comprising water isparticularly advantageous when stabilizing blood samples, as hemolysisis reduced during the storage period.

The present invention also provides a mixture comprising the stabilizingcomposition according to the third aspect of the invention mixed with acell-containing biological sample. Suitable and preferred examples ofcell-containing biological samples as well as suitable concentrations ofthe stabilizing agent(s) when mixed with the cell-containing biologicalsample are described above inter alia in conjunction with thestabilizing method according to the invention. It is referred to theabove disclosure which also applies here. As described, preferably thecell-containing sample is a blood sample.

According to one embodiment, the stabilizing composition of theinvention is pre-filled in a sample collection device so that the sampleis immediately stabilized upon or during collection. According to oneembodiment, the stabilizing composition is contacted with thecell-containing biological sample in a volumetric ratio selected from10:1 to 1:20, 5:1 to 1:15, 1:1 to 1:10, 1:10 to 1:5 and 1:7 to 1:5, inparticular about 1:6. It is a particular advantage of the stabilizingcomposition of the present invention that stabilization of a largesample volume can be achieved with a small volume of the stabilizingcomposition. Therefore, preferably, the ratio of stabilizing compositionto sample lies in a range from 1:10 to 1:5, in particular 1:7 to 1:5,such as e.g. about 1:6.

The stabilizing composition according to the third aspect of the presentinvention can be used to stabilize the extracellular nucleic acidpopulation comprised in a cell-containing sample, such as preferably ablood sample. Furthermore, as described above, the stabilizingcomposition stabilizes the contained cells and thereby inter alia reducethe release of genomic DNA and other intracellular nucleic acids fromcells comprised in the cell-containing biological sample. Thereby, acontamination of the extracellular nucleic acid population with genomicDNA and other intracellular nucleic acids is reduced.

The stabilizing composition of the present invention may also beincorporated into a sample collection device, in particular a bloodcollection assembly, such as a blood collection container therebyproviding for a new and useful version of such a device. Such devicestypically include a container having an open and a closed end. Thecontainer is preferably a blood collection tube. The container type alsodepends on the sample to be collected, other suitable formats aredescribed below.

D. Use

According to fourth aspect, the present invention is directed to the useof a poly(oxyethylene) polymer, which preferably is a polyethyleneglycol, and/or the use of mono-ethylene glycol for stabilizing acell-containing biological sample and in particular the extracellularnucleic acid population comprised in a cell-containing biologicalsample. In particular, the stabilizing composition according to thethird aspect can be used for said purpose, e.g. in the method accordingto the first aspect of the present invention. Details of said methodwere described above and it is referred to the above disclosure whichalso applies here. Preferably, as described above, the compositioncomprises an anticoagulant if the cell-containing biological sample isblood what is a preferred embodiment.

E. Collection Device

According to a fifth aspect, the present invention provides a collectiondevice for collecting a cell-containing biological sample, wherein thecollection device comprises

-   -   i) a poly(oxyethylene) polymer as stabilizing agent or    -   ii) mono-ethylene glycol as stabilizing agent        and one or more, preferably two or more, further additives        selected from the group consisting of    -   one or more primary, secondary or tertiary amides;    -   a caspase inhibitor;    -   an anticoagulant and/or a chelating agent.

Preferably, the collection device according to the fifth aspectcomprises a poly(oxyethylene) polymer, which preferably is a highmolecular weight poly(oxyethylene) polymer having a molecular weight ofat least 1500, as stabilizing agent and furthermore comprises one ormore, preferably two or more further additives selected from the groupconsisting of

-   -   at least one further poly(oxyethylene) polymer having a        molecular weight that is at least 100, preferably at least 200,        at least 300 or at least 400 below the molecular weight of the        first poly(oxyethylene) polymer which preferably is a high        molecular weight poly(oxyethylene) polymer, wherein said further        poly(oxyethylene) polymer preferably is a low molecular weight        poly(oxyethylene) polymer having a molecular weight of 1000 or        less;    -   one or more primary, secondary or tertiary amides;    -   a caspase inhibitor;    -   an anticoagulant and/or a chelating agent.

Providing a respective collection device, e.g. a sample collection tube,has the advantage that the sample is quickly stabilized when the sampleis collected in said collection device. The collection device forcollecting a cell-containing biological sample, preferably a bloodsample, may comprise a stabilizing composition according to the thirdaspect of the present invention. Details with respect to the the use ofa poly(oxyethylene) polymer and optionally the one or more furtheradditives for stabilization as well as the stabilizing composition weredescribed above in conjunction with the other aspects, it is referred tothe above disclosure which also applies here. The same applies withrespect to the alternative wherein mono-ethylene glycol is used asstabilizing agent, details of that embodiment were described alreadyabove. Mono-ethylene glycol can also be used in combination with the atleast one poly(oxyethylene)polymer. The collection device issubsequently also referred to as container.

According to one embodiment, the collection device comprises apoly(oxyethylene) polymer, preferably a polyethylene glycol, one or moreprimary, secondary or tertiary amides, preferably one or more compoundsaccording to formula 1 and a caspase inhibitor. Furthermore, if thecollection container is for collection blood, it preferably alsocomprises an anticoagulant, which preferably is a chelating agent.

According to one embodiment, a collection device for receiving andcollecting a cell-containing biological sample is provided whichcomprises:

-   -   a) at least one high molecular weight poly(oxyethylene) polymer        having a molecular weight of at least 1500, at least 2000,        preferably at least 3000, wherein the molecular weight        preferably lies in a range selected from 2000 to 40000, 2500 to        30000, 2500 to 25000, 3000 to 20000, 3500 to 15000, 4000 to        10000, 4500 to 8000 and 5000 to 7000;    -   b) one or more primary, secondary or tertiary amides;    -   c) at least one caspase inhibitor, preferably a pancaspase        inhibitor, more preferred Q-VD-OPh;    -   d) optionally at least one low molecular weight        poly(oxyethylene) polymer having a molecular weight of 1000 or        less, wherein the molecular weight preferably lies in a range        selected from 100 to 800, 150 to 700, 150 to 600, 200 to 500 and        200 to 400;    -   e) optionally an anticoagulant and/or chelating agent, more        preferably EDTA.

In this embodiment, the high molecular weight poly(oxethylene) polymer(component a)) which preferably is a polyethylene glycol, may becomprised in the collection device in a concentration so that when thecell-containing biological sample is collected into said device, theconcentration of the high molecular weight poly(oxyethylene) polymer inthe resulting mixture lies in a range selected from 0.05% to 4% (w/v),0.1% to 3% (w/v), 0.2% to 2.5% (w/v), 0.25% to 2% (w/v), 0.3% to 1.75%(w/v) and 0.35% to 1.5% (w/v). According to one embodiment, the highmolecular weight poly(oxyethylene) polymer is comprised in thecollection device in a concentration so that when the cell-containingbiological sample is collected into said device, the concentration ofthe high molecular weight poly(oxyethylene) polymer in the resultingmixture lies in a range as 0.25% to 1.5% (w/v), 0.3% to 1.25% (w/v),0.35% to 1% (w/v) and 0.4% to 0.75% (w/v). These concentrations rangesare particularly suitable for the stabilization of blood and theadvantages were discussed above in conjunction with the stabilizationmethod.

In this embodiment, the one or more primary, secondary or tertiary amide(component b)) is comprised in the collection device in a concentrationso that when the cell-containing biological sample is collected intosaid device, the concentration of the amide (or combination of amides)in the resulting mixture lies in a range of 0.25% to 5%, 0.3% to 4%,0.4% to 3%, 0.5% to 2% or 0.75% to 1.5%. The at least one amidepreferably is a compound according to formula 1

wherein R1 is a hydrogen residue or an alkyl residue, preferably a C1-C5alkyl residue, a 01-C4 alkyl residue or a C1-C3 alkyl residue, morepreferred a C1-C2 alkyl residue, R2 and R3 are identical or differentand are selected from a hydrogen residue and a hydrocarbon residue,preferably an alkyl residue, with a length of the carbon chain of 1-20atoms arranged in a linear or branched manner, and R4 is an oxygen,sulphur or selenium residue. Preferably, the amide is a carboxylic acidamide so that R4 is oxygen. Preferred embodiments of the compoundaccording to formula 1 are described above in conjunction with thestabilization method and it is referred to the above disclosure whichalso applies here. Preferably, the collection device comprisesbutanamide and/or a an N,N-dialkylpropanamide which preferably isN,N-dimethylproanamide as compound according to formula 1.

In this embodiment, the at least one caspase inhibitor (component c)) isadvantageously comprised in the collection device in a concentration sothat when the cell-containing biological sample is collected into saiddevice, the concentration of the caspase inhibitor in the resultingmixture lies in a range of 0.1 μM to 20 μM, more preferred 0.5 μM to 10μM, more preferred 1 μM to 10 μM, more preferred 3 μM to 7.5 μM or 3 μMto 5 μM. As is shown by the examples, a stabilizing compositioncomprising a poly(oxyethylene) polymer and a caspase inhibitor is veryeffective in stabilizing a cell-containiner biological sample, inparticular a whole blood sample. Preferably, one or more primary,secondary or tertiary amides as described above are additionally used.

If in this embodiment the collection device additionally comprises a lowmolecular weight poly(oxyethylene) polymer having a molecular weight of1000 or less as component d), it is advantageously comprised in thecollection device in a concentration so that when the cell-containingbiological sample is collected into said device, the concentration oflow molecular weight poly(oxyethylene) polymer in the resulting mixturelies in a range selected from 0.5% to 10%, 1.5% to 9%, 2% to 8% and 2.5%to 7% and 3% to 6%. The percentage values refer to (w/v) in case thepoly(oxyethylene) polymer is a solid and to (v/v) in case thepoly(oxyethylene) polymer is a liquid. Preferably, the poly(oxyethylene)polymer is a polyethylene glycol. The advantages associated with thisembodiment, wherein a high molecular weight poly(oxyethylene) polymer isused in combination with a low molecular weight poly(oxyethylene)polymer was described in detail above. In an advantageous embodiment,the collection container comprises the high molecular weightpoly(oxyethylene) polymer (component a)) and the low molecular weightpoly(oxyethylene) polymer (component d)) in a concentration so that whenthe cell-containing biological sample is collected into said device, theconcentration of the high molecular weight poly(oxyethylene) polymer inthe resulting mixture lies for the high molecular weightpoly(oxyethylene glycol in a range selected from 0.2% to 1.5% (w/v),0.3% to 1.25% (w/v) and 0.4% to 0.75% (w/v) and for the low molecularweight poly(oxyethylene) glycol in a range selected from 1.5% to 8%, 2%to 7% and 2.5% to 6%. Preferably, the high as well as the low molecularweight poly(oxyethylene) polymer is a polyethylene glycol as describedabove.

According to one embodiment, the collection device additionallycomprises an anticoagulant and/or a chelating agent agent. Thisembodiment is particularly suitable if the container is for collectingblood or a sample derived from blood such as plasma or serum. Theanticoagulant is comprised in a concentration wherein it is capable ofpreventing the coagulation of blood. Suitable anticoagulants weredescribed above in conjunction with the method according to the firstaspect and it is referred to the above disclosure which also applieshere. As described, the anticoagulant is preferably a chelating agentsand suitable embodiments were described in detail above and it isreferred to the respective disclosure. According to one embodiment, thecontainer comprises a chelating agent, preferably EDTA, in aconcentration so that when the cell-containing biological sample iscollected into the container, the concentration of the chelating agentin the resulting mixture lies in a concentration range selected from 0.5to 40 mg/ml; 1 to 30 mg/ml, 1.6 to 25 mg/ml, 5 to 20 mg/ml and 7.5 to17.5 mg/ml.

According to one embodiment, the collection device comprisesmono-ethylene glycol as stabilizing agent. Mono-ethylene glycol may beused in the concentrations described above for the low molecular weightpoly(oxyethylene) polymer. It is referred to the repective disclosurewhich also applies here. In particular, the collection device maycomprise mono-ethylene glycol and any one or more of the otherstabilizing agents described herein, preferably at least one caspaseinhibitor and/or at least one primary, secondary or tertiary amide,which preferably is a compound according to formula 1 as defined above.The collection device may also comprise mono-ethylene glycol and atleast one poly(oxyethylene) polymer. Suitable embodiments andconcentration ranges for the respective stabilizing agents are describedabove and also apply to the embodiment, wherein mono-ethylene glycol iscomprised in the collection container in order to stabilize or supportthe stabilization of a cell-containing sample and the exracellularnucleic acid population comprised therein.

The stabilizing composition and/or the individual compounds used forstabilization comprised in the collection container can be provided in aliquid; semi-liquid or in a dry form. As discussed above, thepoly(oxyethylene) polymer and the further additives used forstabilization may be provided in form of a stabilizing composition. Thecompounds used for stabilization may also be provided as separateentities in the container and may also be provided in different forms inthe container. E.g. one component may be provided in dry form while theother compound may be provided as liquid. Other combinations are alsofeasible. Suitable formulation and manufacturing options are known tothe skilled person.

For stabilizing whole blood it is preferred to encompass ananticoagulant such as EDTA into the container, e.g. in the stabilizingcomposition. A dry form is e.g. a suitable option if the biologicalsample to be stabilized contains liquid to dissolve the solid (such asfor example cell-containing body fluids, cells in medium, urine) or ifliquid, e.g. water or other solvent is added thereto to dissolve thesolid. The advantage of using a solid stabilizing composition is thatsolids are usually chemically more stable than liquids. According to oneembodiment, the inner wall of the container is treated/covered with astabilizing composition according to the present invention or withindividual components thereof, such as e.g. the anticoagulant. Saidcomposition or component can be applied to the inner walls using e.g. aspray-dry-method. Liquid removal techniques can be performed on thestabilizing composition in order to obtain a substantially solid stateprotective composition. Liquid removal conditions may be such that theyresult in removal of at least about 50% by weight, at least about 75% byweight, or at least about 85% by weight of the original amount of thedispensed liquid stabilizing composition. Liquid removal conditions maybe such that they result in removal of sufficient liquid so that theresulting composition is in the form of a film, gel or othersubstantially solid or highly viscous layer. For example it may resultin a substantially immobile coating (preferably a coating that can bere-dissolved or otherwise dispersed upon contact with thecell-containing sample which preferably is a blood product sample). Itis possible that lyophilization or other techniques may be employed forrealizing a substantially solid form of the protective agent (e.g., inthe form of one or more pellet). Thus, liquid removal conditions may besuch that they result in a material that upon contact with the sampleunder consideration (e.g., a whole blood sample) the protective agentwill disperse in the sample, and substantially preserve components(e.g., extracellular nucleic acids) in the sample. Liquid removalconditions may be such that they result in a remaining composition thatis substantially free of crystallinity, has a viscosity that issufficiently high that the remaining composition is substantiallyimmobile at ambient temperature; or both.

According to one embodiment, a liquid composition is used. This hasadvantages for specific samples such as e.g. blood samples. Liquidcompositions have the advantage that the mixture with thecell-containing biological sample to be stabilised can be quicklyachieved, thereby basically providing an immediate stabilizing effect assoon as the sample comes into contact with the liquid stabilizingcomposition. Furthermore, liquid compositions are advantageous if largeramounts of stabilization compositions are used which accordingly, cannot or are difficult to spray-dry or if the composition hampersproviding a dry composition. Preferably, the stabilizing agents presentin the liquid stabilizing composition remain stable in solution andrequire no pre-treatment—such as for example the dissolving ofprecipitates of limited solubility—by the user because pre-treatments ofthis kind pose a risk of variations in the stabilizing efficiency. Forstabilizing blood, according to one embodiment, all compounds arepresent in the stabilizing composition. As discussed above, in case ofblood it is advantageous to use a stabilization composition comprising asufficient amount of water to reduce hemolysis during storage of thestabilized sample.

The stabilizing composition is comprised in the container in an amounteffective to provide the stabilization of the amount of sample to becollected in said container. According to one embodiment, the liquidstabilizing composition is contacted with the biological sample in avolumetric ratio selected from 10:1 to 1:20, 5:1 to 1:15, 1:1 to 1:10,1:10 to 1:5 and 1:7 to 1:5, in particular about 1:6. It is a particularadvantage of the stabilizing composition of the present invention thatstabilization of a large sample volume can be achieved with a smallvolume of the stabilizing composition. Therefore, preferably, the ratioof stabilizing composition to sample lies in a range from 1:10 to 1:5,in particular 1:7 to 1:5, such as about 1:6.

According to one embodiment, the collection device is evacuated. Theevacuation is preferably effective for drawing a specific volume of afluid cell-containing biological sample into the interior. Thereby, itis ensured that the correct amount of sample is contacted with thepre-filled amount of the stabilizing composition comprised in thecontainer, and accordingly, that the stabilization is efficient.According to one embodiment, the container comprises a tube having anopen end sealed by a septum. E.g. the container is pre-filled with adefined amount of the stabilizing composition either in solid or liquidform and is provided with a defined vacuum and sealed with a septum. Theseptum is constructed such that it is compatible with the standardsampling accessories (e.g. cannula, etc.). When contacted with e.g. thecanula, a sample amount that is predetermined by the vacuum is collectedin the container. A respective embodiment is in particular advantageousfor collecting blood. A suitable container is e.g. disclosed in U.S.Pat. No. 6,776,959.

The container according to the present invention can be made of glass,plastic or other suitable materials. Plastic materials can be oxygenimpermeable materials or may contain an oxygen impermeable layer.Alternatively, the container can be made of air-permeable plasticmaterial. The container according to the present invention preferably ismade of a transparent material. Examples of suitable transparentthermoplastic materials include polycarbonates, polyethylene,polypropylene and polyethyleneterephthalate. The container may have asuitable dimension selected according to the required volume of thebiological sample being collected. As described above, preferably, thecontainer is evacuated to an internal pressure below atmosphericpressure. Such an embodiment is particularly suitable for collectingbody fluids such as whole blood. The pressure is preferably selected todraw a predetermined volume of a biological sample into the container.In addition to such vacuum tubes also non-vacuum tubes, mechanicalseparator tubes or gel-barrier tubes can be used as sample containers,in particular for the collection of blood samples. Examples of suitablecontainers and capping devices are disclosed in U.S. Pat. No. 5,860,397and US 2004/0043505. As container for collecting the cell-containingsample also further collection devices, for example a syringe, a urinecollection device or other collection devices can be used. The type ofthe container may also depend on the sample type to be collected andsuitable containers are also available to the skilled person.

According to one embodiment, the container has an open top, a bottom,and a sidewall extending therebetween defining a chamber, wherein thepoly(oxyethylene) polymer, which preferably is a high molecular weightpoly(oxyethylene) polymer and the one or more further stabilizing agentsmentioned above or the stabilization composition according to the thirdaspect is comprised in the chamber. It may be comprised therein inliquid or solid form. According to one embodiment, it is a liquid.According to one embodiment the container is a tube, the bottom is aclosed bottom, the container further comprises a closure in the opentop, and the chamber is at a reduced pressure. The advantages of areduced pressure in the chamber were described above. Preferably, theclosure is capable of being pierced with a needle or cannula, and thereduced pressure is selected to draw a specified volume of a liquidsample into the chamber. According to one embodiment, the chamber is ata reduced pressure selected to draw a specified volume of a liquidsample into the chamber, and the stabilizing composition is a liquid andis disposed in the chamber such that the volumetric ratio of thestabilizing composition to the specified volume of the cell-containingsample is selected from 10:1 to 1:20, 5:1 to 1:15 and 1:1 to 1:10 andpreferably is 1:10 to 1:5, more preferably 1:7 to 1:5. The associatedadvantages were described above.

Preferably, the container is for drawing blood from a patient. Accordingto one embodiment, it is for drawing 10 ml blood from a patient.According to one embodiment, the stabilisation composition is a liquidand the volume is 2 ml or less and may lie in a range of 0.5 ml to 2 ml,0.75 ml to 1.75 ml and 1 ml to 1.5 ml.

F. Method for Collecting a Cell Containing Sample

According to a sixth aspect, a method is provided comprising the step ofcollecting a cell-containing biological sample from a patient directlyinto a chamber of a container according to the fifth aspect of thepresent invention. Details with respect to the container and thecell-containing biological sample were described above. It is referredto the respective disclosure. According to one embodiment, a bloodsample is collected, preferably it is drawn from the patient into thecontainer.

G. Manufacturing Method

According to a seventh aspect, a method of manufacturing a stabilizingcomposition according to the third aspect of the present invention isprovided, wherein the components of the stabilizing composition aremixed. Preferably, they are mixed to provide a liquid solution. Asdescribed, a stabilization composition comprising water is particularlypreferred for stabilizing blood samples, because hemolysis can beeffectively reduced.

This invention is not limited by the exemplary methods and materialsdisclosed herein, and any methods and materials similar or equivalent tothose described herein can be used in the practice or testing ofembodiments of this invention. Numeric ranges are inclusive of thenumbers defining the range. The headings provided herein are notlimitations of the various aspects or embodiments of this inventionwhich can be read by reference to the specification as a whole.

Unless the context indicates otherwise, percentage values indicatedherein refer to (w/v) in case of solid compounds contained in a liquidmixture or composition and to (v/v) in case of liquid compoundscontained in a liquid mixture or composition such as e.g. the mixtureresulting from contacting the stabilizing agents or the stabilizingcomposition containing said agents with the cell-containing sample.

As used in the subject specification, the singular forms “a”, “an” and“the” include plural aspects unless the context clearly dictatesotherwise. Thus, for example, reference to “a poly(oxyethylene) polymer”includes a single type of poly(oxyethylene) polymer, as well as two ormore poly(oxyethylene) polymers. Likewise, reference to an “agent”,“additive”, “compound” and the like includes single entities andcombinations of two or more of such entities. Reference to “thedisclosure” and “the invention” and the like includes single or multipleaspects taught herein; and so forth. Aspects taught herein areencompassed by the term “invention”.

The term “solution” as used herein in particular refers to a liquidcomposition, preferably an aqueous composition. It may be a homogenousmixture of only one phase but it is also within the scope of the presentinvention that a solution comprises solid additives such as e.g.precipitates, in particular of contained chemicals such as stabilizingagents.

The sizes, respectively size ranges indicated herein with reference tonucleotides (nt), refer to the chain length and thus are used in orderto describe the length of single-stranded as well as double-strandedmolecules. In double-stranded molecules said nucleotides are paired.

According to one embodiment, subject matter described herein ascomprising certain steps in the case of methods or as comprising certainingredients in the case of compositions, solutions and/or buffers refersto subject matter consisting of the respective steps or ingredients. Itis preferred to select and combine preferred embodiments describedherein and the specific subject-matter arising from a respectivecombination of preferred embodiments also belongs to the presentdisclosure.

The present application claims priority of EP 14 000 990.3 and U.S.61/955,200 (filed: Mar. 18, 2014), the disclosure of both applicationsis herewith incorporated by reference.

EXAMPLES

It should be understood that the following examples are for illustrativepurpose only and are not to be construed as limiting this invention inany manner.

ABBREVIATIONS USED

-   BA: Butanamide-   ccfDNA: circulating, cell free DNA-   DMPA: Dimethylpropionamide-   EDTA: Ethylenediaminetetraacetic acid-   PEG: Polyethylene glycol

Polyethylene glycol (PEG) was tested for its ability to stabilize acell-containing biological sample, here a blood sample, either alone orin combination with different stabilizing agents, including a caspaseinhibitor and/or different primary and/or tertiary amides. Compared tothe reference samples (EDTA blood), PEG was found to be able toefficiently stabilize white blood cells in whole blood samples in a way,that it prevents the release of genomic DNA into the extracellularnucleic acid population. This stabilization effect was demonstrated forPEG of different molecular weights and when used in differentconcentrations. It is demonstrated that PEG can be added as a water-freepowder or liquid, as pure reagent or dissolved in an aqueous solution.PEG alone has a strong stabilization effect on its own, but it alsosignificantly improves the stabilization in combination with otherstabilizing agents, including caspase inhibitors and different amides toa level that the increase of DNA released from white blood cells intoplasma between day 0 (directly after blood draw) and day 6 (6 days ofstorage at room temperature) is reproducible reduced to 2 fold or evenlower. This achieved prolonged, efficient stabilization is an importantadvantage, as it provides a uniform, reliable stabilization method forcell-containing samples such as blood samples. Furthermore, the examplesdemonstrate that it is advantageous to use a combination of a highmolecular weight PEG and a low molecular weight PEG as strongstabilizing effects are achieved and extracellular nucleic acids can beefficiently isolated from stabilized samples using e.g. silica columnbased nucleic acid isolation methods.

I. MATERIALS AND METHODS

The following procedure was followed in the examples if not indicatedotherwise.

1. Blood Collection and Stabilization

Blood was drawn into 10 ml spray dried EDTA tubes (BD) with 1.8 mgK2EDTA per ml of whole blood. Within 30 min after draw, stabilizationreagents were either directly added or the blood was decanted into a newtube containing stabilization reagents. Blood and reagents were mixed byinverting the tube ten times. Stabilized blood samples were stored atroom temperature standing in an upright position.

2. Preparation of Plasma

Whole blood samples were centrifuged at ambient temperature for 15 minat 3.000 rpm (resolutions per minute). Clear plasma fraction was removedby pipetting and transferred into a fresh centrifuge tube. In a secondround, plasma samples were centrifuged at 4° C. for 10 min at 16.000×g.Supernatant was transferred into a new tube and either directly used forpurification of ccfDNA or stored at −20° C. until use.

3. Purification of ccfDNA

DNA from plasma was isolated with the QIAamp circulating nucleic acidkit (QIAGEN GmbH), using the protocol for “purification of circulatingnucleic acids from 1 ml, 2 ml, or 3 ml serum or plasma”. If not statedotherwise, 2 ml of plasma was mixed with proteinase K and lysis bufferACL, incubated for 30 min at 60° C., mixed with buffer ACB, bound onQIAamp Mini columns (which comprise a silica solid phase for binding thenucleic acids) with the use of a QIAvac 24 Plus vacuum manifold, washedand eluted with 60 μl elution buffer AVE, according to the manufacturesrecommendations.

4. Quantitative, Real Time PCR Assay for Analyzing the IsolatedExtracellular DNA

The isolated extracellular DNA was analysed in a real time PCR assay onAbi Prism HT7900 (Life technologies) using 3 μl of eluate. In a 20 μlassay volume using QuantiTect Multiplex PCR Kit reagents (QIAGEN GmbH)two fragments of the human 18S rDNA gene, 66 bp and 500 bp, wereamplified in a multiplex PCR. Cycle threshholds (Ct values) of theindividual samples were translated into amount of gDNA in the eluateaccording to a gDNA standard curve: total quantification was achieved bycomparison with a standard curve generated with human genomic DNAdiluted from 3000 to 0.3 genome equivalents (1 genome equivalent equatesto around 6.6 pg of human genomic DNA). The gDNA amount of the storagetime point (in general 6 days after blood withdrawal) was compared tothe time zero gDNA level from the same donor.

TABLE 1 summarizes the information ofthe used DNA target sequences detected by quantitative real time PCRTarget de- Sequence scription position position 5′-3′ dye h 18 S p12- Forward GCCGCTAG 5′ Cy5- rDNA region of AGGTGAAA BHQ 3′  66 bpchromosome TTCTTG amplicon 13, 14, reverse CATTCTTG 15, 21, GCAAATGC 22TTTCG probe ACCGGCGC AAGACGGA CCAGA h 18 S p12- forward GTCGCTCG 5′ FAM-rDNA region of CTCCTCTC BHQ 3′ 500 bp chromosome CTACTT amplicon 13, 14,reverse GGCTGCTG 15, 21, GCACCAGA 22 CTT probe CTAATACA TGCCGACGGGCGCTGA C

Quantification of the 66 bp fragment was used to deflect the totalamount of 18S rDNA copies in the plasma. Quantification of the 500 bpwas used to determine the amount of 18S rDNA copies which derived fromapoptotic or mechanically lysed leucocytes from whole blood. Cell freeDNA has a typically lengths of 140-170 bp. Therefore, 500 bp fragmentsare believed to be derived from apoptotic, lysed or otherwise destructedblood cells. The increase of copy numbers from the 500 bp fragmentbetween T0 and 6 days storage, was used as a measurement of stabilityefficiency. Thus, the lower the amount of released 500 bp DNA, thebetter the performance of the stabilization method. A higher amount ofreleased 500 bp DNA indicates that lysis of white blood cells occurs andhence, that the extracellular nucleic acid population was contaminatedwith intracellular genomic DNA.

For the subsequent experiments with different stabilization compositionsblood samples from a plurality of different individual donors were used.The average fold change of copy numbers of 66 bp and 500 bp fragments ofthe 18S rDNA gene in stabilized or unstabilized blood stored fordifferent time points (days) at room temperature to time point 0 (day 0)after blood draw was single calculated for each individual donor sample.The average of the corresponding single calculated mean values (foldchanges) was used as a measure of stabilization efficacy of thedifferent stabilization compositions used. As blood samples underlienatural individual variations in their composition and in the amount ofcontained extracellular nucleic acids depending on the donor, this mayresult in elevated standard deviations.

5. Measurement of Haemoglobin

Absorbance at 414 nm, found to be linearly correlated with hemolyticaldiscoloration of plasma, was measured on a spectramax photometer.

II. EXAMPLES 1. Example 1

In example 1, the stabilization effect of polyethylene glycol (PEG) withdifferent molecular weights in combination with BA, EDTA and a caspaseinhibitor (Q-VD-OPh) in the absence of water was tested and compared toa combined BA, EDTA and caspase inhibitor (Q-VD-OPh) approach. Moreover,the effect on hemolysis of such stabilization mixtures in plasma sampleswas measured in parallel. An EDTA blood sample served as unstabilizedreference control.

Blood Collection and Stabilization

Samples of 10 ml whole blood from eight donors, collected in 10 ml spraydry K2EDTA tubes, were stabilized with mixtures of butanamide (BA) andEDTA with or without PEG from different molecular weights withoutaddition of water. Caspase inhibitor (Q-VD-OPh) dissolved in DMSO wasadded by pipetting. Plasma was directly generated from 5 ml ofstabilized or unstabilized blood samples. Residual blood was stored foradditional 6 days at room temperature before plasma generation. ccfDNAwas purified from 2 ml plasma, copy numbers of 18S rDNA gene weredetermined in triplicates by real time PCR.

All stabilized blood samples were set up in triplicates per conditionand test time point. At time point 0 (reference time point), immediatelyafter mixing the stabilization solution and blood, plasma was generatedand the circulating extracellular DNA was extracted. As a referencecontrol, the EDTA stabilized blood sample (collected in K2 EDTA tubeswithout further additives) was also stored for 0 or 6 days and analysedin triplicates.

Composition of stabilization reagent mixtures (for 10 ml K2EDTA wholeblood each):

-   -   unstabilized: 1.8 mg/ml K2EDTA    -   BA, EDTA, Q-VD-OPh: 100 mg BA, 132 mg K2EDTA, 10 μl Q-VD-OPh (1        mg dissolved in 388 μl DMSO), water ad 2 ml    -   PEG (600, 1000 or 3000), BA, EDTA, Q-VD-OPh: 250 mg PEG (600,        1000 or 3000), 100 mg BA, 132 mg K2EDTA, 10 μl Q-VD-OPh (1 mg        dissolved in 388 μl DMSO) (no water)

Thereby, the following final concentrations of the different componentsin the mixture are obtained after contact with blood:

-   -   unstabilized: 1.8 mg/ml K2EDTA    -   BA, EDTA, Q-VD-OPh: 1% (w/v) BA, 15 mg/ml K2EDTA, 5 μM Q-VD-OPh    -   PEG (600, 1000 or 3000), BA, EDTA, Q-VD-OPh: 2.5% (w/v) PEG        (600, 1000 or 3000), 1% (w/v) BA, 15 mg/ml K2EDTA, 5 μM Q-VD-OPh

Results

The average change of copy numbers (x fold change) of 66 bp and 500 bpfragments of the 18S rDNA gene in stabilized or unstabilized blood from8 donors stored for 6 days at room temperature to time point 0 (day 0)after blood draw was single calculated for each of the eight blooddonors. FIG. 1 shows the corresponding average fold change of copynumbers from 8 donors per condition. All stabilization compositions showsignificantly lower amounts of released genomic DNA after storage for 6days at room temperature compared to the unstabilized control (EDTAblood) as the average fold change increases significantly less. Thus, astabilization effect was achieved even throughout this longstabilization period of 6 days. FIG. 1 demonstrates that contacting theblood samples additionally with polyethylene glycol significantlyimproved the stabilization effect achieved. Therefore, PEG of differentmolecular weights were highly effective in improving the stabilizationeffect as the average fold change increase was consistently reducedbelow 2-fold and in embodiments even below 1-fold. I.e. the DNA levelsat day 6 are comparable to that of the basal time point (day 0).

To summarize, the stabilization effect of a stabilization compositioncomprising a caspase inhibitor and an amide, here a primary carboxylicacid amide, can be significantly increased when used in combination witha polyethylene glycol. Polyethylene glycol was effective in differentmolecular weights. Moreover, the results indicate that the stabilizationproperties of PEG increased with increasing molecular weight of PEG,indicating that there is a positive correlation between the molecularweight of used PEG and the resulting sample stabilization effect. Highermolecular weights improved the achieved stabilization effect.

2. Example 2

In example 2, the stabilization effect of a combination of EDTA, BA anda caspase inhibitor (Q-VD-OPh) in the absence of water was tested andcompared to corresponding compositions additionally including differentamounts (0.2 g, 0.3 g or 0.4 g) of PEG with a molecular weight of 600(PEG600). EDTA blood served as unstabilized reference.

Blood Collection and Stabilization

Samples of 10 ml whole blood from six donors, collected in 10 ml spraydry K2EDTA tubes, were stabilized with mixtures of butanamide (BA) andEDTA with or without different amounts of PEG with a molecular weight of600 (PEG600) without addition of water. In addition, a caspase inhibitor(Q-VD-OPh) dissolved in DMSO was added by pipetting. Plasma was directlygenerated from 5 ml of stabilized or unstabilized blood samples.Residual blood was stored for additional 6 days at room temperaturebefore plasma generation. ccfDNA was purified from 2 ml plasma, copynumbers of 18S rDNA gene were determined in triplicates per conditionand test time point by real time PCR. As a reference control, the EDTAstabilized blood sample (collected in K2 EDTA tubes without furtheradditives) was also stored for 6 days.

Composition of stabilization reagent mixtures (for 10 ml K2EDTA wholeblood each):

-   -   BA, EDTA, Q-VD-OPh: 100 mg BA, 182 mg K2EDTA, 10 μl Q-VD-OPh (1        mg dissolved in 388 μl DMSO), ad 2 ml water    -   PEG600 (0.2-0.4 g), BA, EDTA, Q-VD-OPh: 200, 300 and 400 mg        PEG600,100 mg BA, 188 mg K2EDTA, 10 μl Q-VD-OPh (1 mg dissolved        in 388 μl DMSO)

Thereby, the following final concentrations of the different componentsin the mixture are obtained after contact with blood:

-   -   unstabilized: 1.8 mg/ml K2EDTA    -   BA, EDTA, Q-VD-OPh: 1% (w/v) BA, 20 mg/ml K2EDTA, 5 μM Q-VD-OPh    -   PEG600 (0.2-0.4 g), BA, EDTA, Q-VD-OPh: 2, 3 and 4% (w/v)        PEG600, 1% (w/v) BA, 20 mg/ml K2EDTA, 5 μM Q-VD-OPh

Results

The results of the quantitative real time PCR analyses from sixindividual donor samples depicted as average fold change is shown inFIG. 2. The increase of DNA (66 bp and 500 bp fragment) relative to timezero with 0.2 g, 0.3 g or 0.4 g PEG600 (average fold change) is shown.All tested stabilization compositions showed significant lower amountsof released DNA after storage for 6 days at room temperature compared tothe reference EDTA blood. The stabilization effect was significantlyimproved if the cell-containing sample was additionally contacted withpolyethylene glycol. The average fold change of both 18S rDNA ampliconcopy numbers was clearly smaller in all three PEG based stabilizationapproaches compared the composition not comprising PEG (BA, EDTA,Q-VD-OPh). The x fold change was in all cases below 2-fold. This exampledemonstrates that additionally using a polyethylene glycol in differentquantities for stabilizing the extracellular nucleic acid populationsignificantly improves the stabilization results that are achieved withthe caspase inhibitor and the primary carboxylic acid amide butanamide.

3. Example 3

In example 3, the stabilization effect of reagent mixtures, including ahigh molecular weight PEG (PEG3000), EDTA, BA and caspase inhibitor(Q-VD-OPh), directly lyophilized into blood collection tubes in thepresence of water was tested and compared to a sample concomitantlytreated with a solution comprising BA, EDTA and a caspase inhibitor(Q-VD-OPh).

Blood Collection and Stabilization

Samples of 10 ml whole blood from eight donors, collected in 10 ml spraydry K2EDTA tubes, were stabilized with mixtures of butanamide (BA),EDTA, and caspase inhibitor (Q-VD-OPh) with or without PEG3000. Forlyophilisation all components including caspase inhibitor, EDTA, BA andPEG were dissolved in water. Volumes of 1 ml (final concentrations seebelow) were lyophilized on a dry freezer Epsilon 2-25D (Christ GmbH) in5 ml tubes. Blood was transferred from K2EDTA tubes into the 5 ml tubeswith the lyophilized stabilization reagent and stabilized by 10 timesinverting the tubes. As a reference, reagents were freshly prepared andcaspase inhibitor (Q-VD-OPh) dissolved in DMSO was added by pipetting.

Plasma was directly generated from 5 ml of stabilized or unstabilizedblood samples. Residual blood was stored for additional 6 days at roomtemperature before plasma generation. ccfDNA was purified from 2 mlplasma, copy numbers of 18S rDNA gene were determined in triplicates byreal time PCR.

Composition of stabilization reagent mixtures (for 10 ml K2EDTA wholeblood each):

-   -   Freshly prepared BA, EDTA, Q-VD-OPh: 100 mg BA, 132 mg K2EDTA,        10 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), ad 2 ml water    -   Freshly prepared PEG3000, BA, EDTA, Q-VD-OPh: 250 mg PEG3000,        100 mg BA, 132 mg K2EDTA, 10 μl Q-VD-OPh (1 mg dissolved in 388        μl DMSO) (no water)

Composition of stabilization reagent mixtures in 0.5 ml forlyophilisation into 5 ml tubes:

-   -   Lyophilized: 0.5 ml of stabilization reagent containing 125 mg        PEG3000, 50 mg BA, 67.5 mg K2EDTA, 5 μl Q-VD-OPh (1 mg dissolved        in 388 μl DMSO)

Thereby, the following final concentrations of the different componentsin the mixture are obtained after contact with blood:

-   -   unstabilized: 1.8 mg/ml K2EDTA    -   BA, EDTA, Q-VD-OPh: 1% (w/v) BA, 15 mg/ml K2EDTA, 5 μM Q-VD-OPh    -   PEG3000, BA, EDTA, Q-VD-OPh: 1% (w/v) PEG 3000, 1% (w/v) BA, 15        mg/ml K2EDTA, 5 μM Q-VD-OPh

Results

The results are shown in FIG. 3. Shown is the increase (average foldchange) of DNA 6 days after blood withdrawal relative to time zero basedon different amplicon length of the 18S rDNA gene. The results againdemonstrate that the stabilization effect is significantly improved ifpolyethylene glycol is additionally used for stabilization and that itenhances the stabilization effect that is achieved with BA, EDTA and acaspase inhibitor (Q-VD-OPh). Also during the prolonged stabilizationperiods tested (6 days), the x fold change was below 2-fold.Furthermore, the example demonstrates that these stabilizationcompositions may be used either freshly prepared or in lyophilized form.

4. Example 4

In example 4, the stabilization effect of PEG6000 (high molecular weightPEG) either alone or in combination with a caspase inhibitor (Q-VD-OPh)on EDTA stabilized blood samples was tested in an aqueous stabilizationsolution and compared to EDTA stabilized blood alone or BA, EDTAstabilized blood.

Blood Collection and Stabilization

Samples of 10 ml whole blood from eight donors, collected in 10 ml spraydry K2EDTA tubes, were stabilized with aqueous solutions containingeither butanamide or PEG in combination with EDTA and optional caspaseinhibitor (Q-VD-OPh). Plasma was directly generated from 5 ml ofstabilized or unstabilized blood samples. Residual blood was stored foradditional 3 days at room temperature before plasma generation. ccfDNAwas purified from 2 ml plasma, copy numbers of 18S rDNA gene weredetermined in triplicates by real time PCR.

Composition of stabilization reagent mixtures (for 10 ml K2EDTA wholeblood each):

-   -   BA, EDTA, Q-VD-OPh: 360 mg BA, 68.4 mg K2EDTA, with or without        2.4 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), water ad 2 ml    -   PEG6000, EDTA, Q-VD-OPh: 137.5 mg PEG6000, 132 mg K2EDTA, with        or without 2.2 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO),        water ad 1 ml

Thereby, the following final concentrations of the different componentsin the mixture are obtained after contact with blood:

-   -   unstabilized: 1.8 mg/ml K2EDTA    -   BA, EDTA: 3% (w/v) BA, 7.2 mg/ml K2EDTA    -   BA, EDTA, Q-VD-OPh: 3% (w/v) BA, 7.2 mg/ml K2EDTA, 1 μM Q-VD-OPh    -   PEG6000, EDTA: 1.25% (w/v) PEG6000, 7.2 mg/ml K2EDTA    -   PEG6000, EDTA, Q-VD-OPh: 1.25% (w/v) PEG6000, 7.2 mg/ml K2EDTA,        1 μM Q-VD-OPh

Results

The results of qPCR analyses from eight different donors are shown inFIG. 4. Shown is the average change of copy numbers (fold change) of DNAcopies of different 18S rDNA gene amplicons (66 bp or 500 bp) instabilized or unstabilized blood from eight donors stored for 3 days atroom temperature relative to time point 0 (day 0) after bloodwithdrawal. The stabilization compositions comprising the high molecularweight polyethylene glycol show significantly lower amounts of releasedDNA after storage for 3 days at room temperature compared to theunstabilized EDTA blood. The stabilization composition comprising onlythe high molecular weight PEG as stabilizer achieved over a three daystorage period a stabilization effect that was better than the effectachieved with the stabilizing agent butanamide. This demonstrates thatpolyethylene glycol is also alone effective as stabilizing agent, if thestabilization is to be achieved over shorter stabilization periods. Whenusing polyethylene glycol in combination with a caspase inhibitor(Q-VD-OPh), the stabilization effect was improved. The stabilizationeffect achieved with a combination of a high molecular weight PEG andthe caspase inhibitor is even superior to a stabilization approach usinga combination of butanamide and a caspase inhibitor. The resultsdemonstrate that PEG dissolved in an aqueous solution in combinationwith only an anticoagulant (EDTA) stabilizes both ccfDNA and also whiteblood cells (thereby preventing the release of cellular DNA into theplasma). Moreover, it was found that this stabilization effect is evenmore pronounced compared to butanamide.

5. Example 5

In example 5, the stabilization effect of PEG with different molecularweights (PEG300, PEG600, PEG1000) in an aqueous stabilization solutionsfurther comprising BA, EDTA and a caspase inhibitor (Q-VD-OPh) wastested and compared to a sample co-treated with BA, dimethylpropionamide(DMPA), EDTA and caspase inhibitor (Q-VD-OPh). Unstabilized EDTA bloodserved as reference control.

Blood Collection and Stabilization

Samples of 10 ml whole blood from eight donors, collected in 10 ml spraydry K2EDTA tubes, were stabilized with mixtures of butanamide, EDTA andcaspase inhibitor (Q-VD-OPh) in an aqueous solution with or without PEGof different molecular weights. Plasma was directly generated from 5 mlof stabilized or unstabilized blood samples. Residual blood was storedfor additional 6 days at room temperature before plasma generation.ccfDNA was purified from 2 ml plasma, copy numbers of 18S rDNA gene weredetermined in triplicates by real time PCR.

Composition of stabilization reagent mixtures (for 10 ml K2EDTA wholeblood each):

-   -   BA, DMPA, EDTA, Q-VD-OPh: 180 mg BA, 180 μl DMPA, 68.4 mg        K2EDTA, 12 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), water ad        2 ml    -   PEG (300, 600 or 1000), BA, EDTA, Q-VD-OPh: 287.5 mg PEG (300,        600 or 1000), 115 mg BA, 154.5 mg K2EDTA, 11.5 μl Q-VD-OPh (1 mg        dissolved in 388 μl DMSO), water ad 1.5 ml

Thereby, the following final concentrations of the different componentsin the mixture are obtained after contact with blood:

-   -   unstabilized: 1.8 mg/ml K2EDTA    -   BA, DMPA, EDTA, Q-VD-OPh: 1.5% (w/v) BA, 1.5% (v/v) DMPA, 7.2        mg/ml K2EDTA, 5 μM Q-VD-OPh    -   PEG (300, 600 or 1000), BA, EDTA, Q-VD-OPh: 2.5% (w/v) PEG, 1%        (w/v) BA, 15 mg/ml K2EDTA, 5 μM Q-VD-OPh

Results

FIG. 5 shows the achieved stabilization results. As can be seen, theaqueous stabilization compositions comprising PEG of different molecularweight stabilized the blood samples and further increased thestabilization effect that was achieved with butanamide and the caspaseinhibitor. The stabilization of white blood cells was significantlyimproved as can be seen from the reduced amount of contaminating genomicDNA. The results also demonstrate that the stabilization effectincreases with increasing molecular weight of the used PEG. The increaseof the 500 bp fragment was reduced below 2-fold with when usingpolyethylene glycol having a molecular weight of 1000.

6. Example 6

Here, the stabilization effect of decreasing PEG concentrations (2%,1.5%, 1% or 0.7%) in an aqueous stabilization solution were tested incombination with butanamide, EDTA and a caspase inhibitor (Q-VD-OPh).Unstabilized EDTA blood served as reference control. A compositioncomprising BA, EDTA and Q-VD-OPh was tested in parallel.

Blood Collection and Stabilization

Samples of 10 ml whole blood from eight donors, collected in 10 ml spraydry K2EDTA tubes, were stabilized with mixtures of butanamide, EDTA andcaspase inhibitor (Q-VD-OPh) in an aqueous solution with or withoutdifferent concentrations of PEG6000. Plasma was directly generated from5 ml of stabilized or unstabilized blood samples. Residual blood wasstored for additional 6 days at room temperature before plasmageneration. ccfDNA was purified from 2 ml plasma, copy numbers of 18SrDNA gene were determined in triplicates by real time PCR.

Composition of stabilization reagent mixtures (for 10 ml K2EDTA wholeblood each):

-   -   BA, EDTA, Q-VD-OPh: 110 mg BA, 147 mg K2EDTA, 11 μl Q-VD-OPh (1        mg dissolved in 388 μl DMSO), water ad 1 ml    -   PEG6000 (2-0.7%), BA, EDTA, Q-VD-OPh: 220, 165, 110, 77 mg        PEG6000, 110 mg BA, 147 mg K2EDTA, 11 μl Q-VD-OPh (1 mg        dissolved in 388 μl DMSO), water ad 1 ml

Thereby, the following final concentrations of the different componentsin the mixture are obtained after contact with blood:

-   -   unstabilized: 1.8 mg/ml K2EDTA    -   BA, EDTA, Q-VD-OPh: 1% (w/v) BA, 15 mg/ml K2EDTA, 5 μM Q-VD-OPh    -   PEG6000 (2-0.7%), BA, EDTA, Q-VD-OPh: 2, 1.5, 1, 0.7% (w/v)        PEG6000, 1% (w/v) BA, 15 mg/ml K2EDTA, 5 μM Q-VD-OPh

Results

In example 6, decreasing concentrations of higher molecular PEG6000 weretested for their influence on the stabilization of white blood cellswhen applied in combination with butanamide, EDTA and a caspaseinhibitor. FIG. 6 depicts the obtained stabilization result of theextracelluar nucleic acid population as determined by analyzing theincrease of 18S rDNA via quantitative real time PCR. All stabilizationcompositions according to the present invention comprising PEG showsignificantly lower amounts of released DNA after storage for 6 days atroom temperature compared to the stabilization approach involvingbutanamide, EDTA and a caspase inhibitor. Moreover, as can be seen, thehigh molecular weight polyethylene glycol can be used in differentconcentrations to stabilize white blood cells in aqueous solutionsthereby reducing contaminations of the extracelluar nucleic acidpopulation with genomic DNA. Furthermore, it is again shown that PEGincreases the stabilization effect of butanamide and the caspaseinhibitor thereby providing a very effective stabilization approach.

7. Example 7

In example 7, the stabilization effect of PEG in an aqueousstabilization solution with different volumes and in combination withEDTA, a caspase inhibitor (Q-VD-OPh) and different amides (BA or DMPA)were tested. Unstabilized EDTA blood served as reference control. Acomposition comprising BA, DMPA, EDTA and Q-VD-OPh was tested inparallel.

Blood Collection and Stabilization

Samples of 10 ml whole blood from eight donors, collected in 10 ml spraydry K2EDTA tubes, were stabilized with mixtures of PEG6000, butanamideor DMPA, EDTA and caspase inhibitor (Q-VD-OPh) in an aqueous solutionwith different volumes 0.8 and 1.2 ml. Plasma was directly generatedfrom 5 ml of stabilized or unstabilized blood samples. Residual bloodwas stored for additional 6 days at room temperature before plasmageneration. ccfDNA was purified from 2 ml plasma, copy numbers of 18SrDNA gene were determined in triplicates by real time PCR.

Composition of stabilization reagent mixtures (for 10 ml K2EDTA wholeblood each):

-   -   BA, DMPA, EDTA, Q-VD-OPh: 180 mg BA, 180 μl DMPA, 68.4 mg        K2EDTA, 12 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), water ad        2 ml    -   PEG6000, BA, EDTA, Q-VD-OPh: 112 mg PEG6000, 56 mg BA, 150 mg        K2EDTA, 2.23 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), water        ad 1.2 or 0.8 ml    -   PEG6000, DMPA, EDTA, Q-VD-OPh: 112 mg PEG6000, 112 μl DMPA, 150        mg K2EDTA, 2.23 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO),        water ad 1.2 or 0.8 ml

Thereby, the following final concentrations of the different componentsin the mixture are obtained after contact with blood:

-   -   unstabilized: 1.8 mg/ml K2EDTA    -   BA, DMPA, EDTA, Q-VD-OPh: 1.5% (w/v) BA, 1.5% (v/v) DMPA, 7.2        mg/ml K2EDTA, 5 μM Q-VD-OPh    -   PEG6000, BA or DMPA, EDTA, Q-VD-OPh ad 1.2 ml: 1% (w/v) PEG6000,        0.5% (w/v) BA or 1% (v/v) DMPA, 15 mg/ml K2EDTA, 1 μM Q-VD-OPh    -   PEG6000, BA or DMPA, EDTA, Q-VD-OPh ad 0.8 ml: 1.04% (w/v)        PEG6000, 0.52% (w/v) BA or 1.04% (v/v) DMPA, 15.6 mg/ml K2EDTA,        1.03 μM Q-VD-OPh

Results

FIG. 7 shows the results of these stabilization assays. Shown is theaverage change of copy numbers (fold change) of DNA copies of different18S rDNA gene amplicons (66 bp or 500 bp) in stabilized or unstabilizedblood stored for 6 days at room temperature relative to time point 0(day 0) after blood withdrawal. The results demonstrate thatpolyethylene glycol can be combined with different amides to stabilizewhite blood cells in different volumes of aqueous solutions, therebyproviding stabilized blood samples wherein the extracellular nucleicacid population is preserved by preventing a dilution with intracellularnucleic acids.

8. Example 8

In example 8, the effect of stabilization reagents in aqueousstabilization solutions is tested by means of hemolysis assays.

Blood Collection and Stabilization

Samples of 10 ml whole blood from eight donors, collected in 10 ml spraydry K2EDTA tubes, were stabilized with mixtures of butanamide or DMPAand EDTA with or without PEG with addition of water. Caspase inhibitor(Q-VD-OPh) dissolved in DMSO was added by pipetting. Plasma was directlygenerated from 5 ml of stabilized or unstabilized blood samples.Residual blood was stored for additional 3, 6 and 10 days at roomtemperature before plasma generation. Hemoglobin content was determinedby measuring absorbance at 414 nm on a spectrophotometer.

Composition of stabilization reagent mixtures (for 10 ml K2EDTA wholeblood each):

-   -   BA, DMPA, EDTA, Q-VD-OPh: 180 mg BA, 180 μl DMPA, 68.4 mg        K2EDTA, 12 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), water ad        2 ml    -   PEG6000, BA, EDTA, Q-VD-OPh: 137.5 mg PEG6000, 55 mg BA, 165 mg        K2EDTA, 11 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), water ad        1.0 ml    -   PEG6000, DMPA, EDTA, Q-VD-OPh: 110 mg PEG6000, 110 μl DMPA, 147        mg K2EDTA, 11 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), water        ad 1.0 ml

Thereby, the following final concentrations of the different componentsin the mixture are obtained after contact with blood:

-   -   unstabilized: 1.8 mg/ml K2EDTA    -   BA, DMPA, EDTA, Q-VD-OPh: 1.5% (w/v) BA, 1.5% (v/v) DMPA, 7.2        mg/ml K2EDTA, 5 μM Q-VD-OPh    -   PEG6000, BA, EDTA, Q-VD-OPh: 1.25% (w/v) PEG6000, 0.5% (w/v) BA,        15 mg/ml K2EDTA, 5 μM Q-VD-OPh    -   PEG6000, DMPA, EDTA, Q-VD-OPh: 1.0% (w/v) PEG6000, 1.0% (v/v)        DMPA, 15 mg/ml K2EDTA, 5 μM Q-VD-OPh

Results

FIG. 8 shows the effect on hemolysis in plasma samples from 8 differentdonors after 0, 3, 6 and 10 days of storage. FIG. 8 depicts the averageincrease of hemolysis from the eight donors as increase of absorbance at414 nm in the plasma fraction.

Whereas in EDTA control experiments hemolysis is elevated over storagetime, hemolysis was reduced in the analyzed time points with the aqueousstabilization solutions according to the invention containing PEG6000combined with EDTA and either BA or DMPA compared to the EDTA referenceas can be seen in FIG. 8. Noteworthy, the increase of hemolysis fromstorage day 6 to storage day 10 seen in the EDTA control is essentiallyreduced in all tested solutions comprising the inventive stabilizationreagent composition and water. The extent of reduced hemolysis in PEGcontaining aqueous solutions was comparable to the combined BA, DMPA,EDTA, caspase inhibitor (Q-VD-OPh) stabilized samples. Therefore,dissolving PEG in aqueous stabilization composition is advantageous asit efficiently reduces hemolysis.

9. Example 9

In example 9 the effect of using different molecular weight PEGs(PEG300, PEG600, PEG1000, PEG3000) on the ccfDNA copy numbers is testedin combination with BA, EDTA and a caspase inhibitor (Q-VD-OPh) andcompared to unstabilized EDTA control blood. A BA, DMPA, EDTA andcaspase inhibitor (Q-VD-OPh) containing composition was tested inparallel.

Blood Collection and Stabilization

Samples of 10 ml whole blood from eight donors, collected in 10 ml spraydry K2EDTA tubes, were stabilized with combinations of butanamide, EDTAand caspase inhibitor (Q-VD-OPh) in an aqueous solution comprising PEGof increasing molecular weights. Plasma was directly generated from 5 mlof stabilized or unstabilized blood samples, one hour after bloodcollection. ccfDNA was purified from 2 ml plasma, copy numbers of 18SrDNA gene were determined in triplicates by real time PCR.

Composition of stabilization reagent mixtures (for 10 ml K2EDTA wholeblood each):

-   -   BA, DMPA, EDTA, Q-VD-OPh: 180 mg BA, 180 μl DMPA, 68.4 mg        K2EDTA, 12 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), water ad        2 ml    -   PEG (300, 600, 1000 or 3000), BA, EDTA, Q-VD-OPh: 287.5 ml PEG        300 or 287.5 mg PEG (600, 1000 or 3000), 115 mg BA, 154.5 mg        K2EDTA, 11.5 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), water        ad 1.5 ml

Thereby, the following final concentrations of the different componentsin the mixture are obtained after contact with blood:

-   -   unstabilized: 1.8 mg/ml K2EDTA    -   BA, DMPA, EDTA, Q-VD-OPh: 1.5% (w/v) BA, 1.5% (v/v) DMPA, 7.2        mg/ml K2EDTA, 5 μM Q-VD-OPh    -   PEG (300, 600, 1000 or 3000), BA, EDTA, Q-VD-OPh: 2.5% (v/v or        w/v) PEG, 1% (w/v) BA, 15 mg/ml K2EDTA, 5 μM Q-VD-OPh

Results

The results of absolute quantitative real time PCR analyses from eightdifferent donors are shown as average in FIG. 9. Particularly shown isthe average of absolute copy numbers of DNA of different 18S rDNA geneamplicons (66 bp or 500 bp) in stabilized or unstabilized blood samplesof the donors at day 0 after blood withdrawal. The aim was to test theeffect of the used stabilization approach on the subsequent nucleic acidyield when using a silica column based nucleic acid isolation procedure.FIG. 9 shows that the addition of a higher molecular weight PEG led to areduction of detectable amplicon gene copy numbers in plasma compared toeither the EDTA reference samples (unstabilized approach) or thestabilized blood solution containing BA, DMPA, EDTA and a caspaseinhibitor (Q-VD-OPh). The results indicates that for the use of PEG withincreasing molecular weight or chain lengths for stabilization may leadwhen used in higher concentrations to a reduction of detectable ccfDNAgene copy numbers in plasma when using a silica column based nucleicacid isolation approach for isolating the extracellular nucleic acidsfrom the stabilized samples.

10. Example 10

In example 10 PEG6000 was tested in different concentrations (1.0%,1.25% or 1.5%) in combination with BA, EDTA and a caspase inhibitor(Q-VD-OPh). An EDTA stabilized blood sample served as reference control.BA, DMPA, EDTA and a caspase inhibitor (Q-VD-OPh) containing bloodmixture was analyzed in parallel.

Blood Collection and Stabilization

Samples of 10 ml whole blood from eight donors, collected in 10 ml spraydry K2EDTA tubes, were stabilized with combinations of amides (DMPAand/or BA), EDTA, caspase inhibitor (0-VD-OPh) with or without PEG6000in a volume of 1.5 ml to 10 ml blood with increasing concentration ofPEG. Plasma was generated from 5 ml of stabilized or unstabilized bloodsamples, one hour after blood collection. ccfDNA was purified from 2 mlplasma, copy numbers of 18S rDNA gene were determined in triplicates byreal time PCR.

Composition of stabilization reagent mixtures for 10 ml K2EDTA wholeblood each:

-   -   BA, DMPA, EDTA, Q-VD-OPh: 180 mg BA, 180 μl DMPA, 68.4 mg        K2EDTA, 12 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), water ad        2 ml    -   PEG6000 (1-1.5%), BA, EDTA, Q-VD-OPh ad 1.5 ml: 115, 144 and 172        mg PEG6000, 115 mg BA, 155 mg K2EDTA, 11.5 μl Q-VD-OPh (1 mg        dissolved in 388 μl DMSO), water ad 1.5 ml

Thereby, the following final concentrations of the different componentsin the mixture are obtained after contact with blood:

-   -   unstabilized: 1.8 mg/ml K2EDTA    -   BA, DMPA, EDTA, Q-VD-OPh: 1.5% (w/v) BA, 1.5% (v/v) DMPA, 7.2        mg/ml K2EDTA, 5 μM Q-VD-OPh    -   PEG6000, BA, EDTA, Q-VD-OPh: 1, 1.25 and 1.5% (w/v) PEG6000, 1%        (w/v) BA, 15 mg/ml K2EDTA, 5 μM Q-VD-OPh

Results

The results of example 10 are shown in FIG. 10. Shown is the averagedecrease of absolute copy numbers of ccfDNA 0 days after blood draw from8 donors based on different amplicon length of the 18S rDNA gene withstabilization compositions according to the invention comprising PEG6000in different concentrations (1.0%, 1.25% or 1.5%) and BA, EDTA and acaspase inhibitor (Q-VD-OPh). FIG. 10 shows that the reduction ofabsolute copy numbers of the 66 bp and 500 bp fragment of the 18S rDNAgene in stabilized blood-plasma containing PEG occurs in a PEGconcentration dependent fashion. This demonstrates that increasingconcentrations of higher molecular PEG (PEG6000) in the stabilizationsolution, may lead to a reduction of detectable ccfDNA gene copy numbersin plasma when using a silica column based nucleic acid isolationapproach.

11. Example 11

In example 11, the stabilization effect of different volumes of anaqueous stabilization composition comprising a high molecular weight PEG(PEG6000) in combination with BA, EDTA and a caspase inhibitor(Q-VD-OPh) was analysed. Blood incubated with a stabilization solutioncomprising a combination of two amides (DMPA, BA), EDTA and a caspaseinhibitor (Q-VD-OPh) was analyzed in parallel. EDTA blood served asunstabilized reference.

Blood Collection and Stabilization

Samples of 10 ml whole blood from eight donors, collected in 10 ml spraydry K2EDTA tubes, were stabilized with combinations of amides (DMPAand/or BA), EDTA, caspase inhibitor (Q-VD-OPh) with or without PEG6000in different volumes of an aqueous solution. Plasma was generated from 5ml of stabilized or unstabilized blood samples, one hour after bloodcollection. ccfDNA was purified from 2 ml plasma, copy numbers of 18SrDNA gene were determined in triplicates by real time PCR.

Composition of stabilization reagent mixtures for 10 ml K2EDTA wholeblood each:

-   -   BA, DMPA, EDTA, Q-VD-OPh: 180 mg BA, 180 μl DMPA, 68.4 mg        K2EDTA, 12 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), water ad        2 ml    -   PEG6000, BA, EDTA, Q-VD-OPh ad 1 ml: 110 mg PEG6000, 110 mg BA,        147 mg K2EDTA, 11 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO),        water ad 1 ml    -   PEG6000, BA, EDTA, Q-VD-OPh ad 1.5 ml: 115 mg PEG6000, 115 mg        BA, 155 mg K2EDTA, 11.5 μl Q-VD-OPh (1 mg dissolved in 388 μl        DMSO), water ad 1.5 ml    -   PEG6000, BA, EDTA, Q-VD-OPh ad 2 ml: 120 mg PEG6000, 120 mg BA,        162 mg K2EDTA, 12 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO),        water ad 2 ml

Thereby, the following final concentrations of the different componentsin the mixture are obtained after contact with blood:

-   -   unstabilized: 1.8 mg/ml K2EDTA    -   BA, DMPA, EDTA, Q-VD-OPh: 1.5% (w/v) BA, 1.5% (v/v) DMPA, 7.2        mg/ml K2EDTA, 5 μM Q-VD-OPh    -   PEG6000, BA, EDTA, Q-VD-OPh ad 1, 1.5 or 2 ml: 1% (w/v) PEG6000,        1% (w/v) BA, 15 mg/ml K2EDTA, 5 μM Q-VD-OPh

Results

In example 11, different volumes of stabilization solutions (1 ml, 1.5ml or 2 ml) according to the invention comprising PEG6000 in combinationwith BA, EDTA and a caspase inhibitor (Q-VD-OPh) were tested. Theresults shown in FIG. 11 demonstrate that the reduction of absolute copynumbers of the two tested fragments of the 18S rDNA gene in thestabilized samples is dependent on the stabilization reagent volume.Thus, increasing the volume of the stabilization composition (andaccordingly increasing the ratio of stabilization composition to blood)containing the high molecular weight PEG lead to a reduction ofdetectable ccfDNA gene copy numbers in plasma. The copy number was notsignificantly reduced when using a lower volume as in apparent from theresults shown for 1 ml stabilization solution. This result issurprising, because the overall concentration was the same in themixture containing the sample.

12. Example 12

Example 12 shows the stabilization effect of stabilization compositionscontaining a combination of a high molecular weight polyethylene glycol(PEG6000) and a low molecular weight polyethylene glycol (PEG300) inaddition to DMPA, EDTA and a caspase inhibitor (0-VD-OPh) in an aqueoussolution with two different volumes (1.5 ml and 2.0 ml). EDTA bloodserves as unstabilized reference control.

Blood Collection and Stabilization

Samples of 10 ml whole blood from eight donors, collected in 10 ml spraydry K2EDTA tubes, were stabilized with mixtures of PEG300 and PEG6000,DMPA, EDTA and caspase inhibitor (Q-VD-OPh) in an aqueous solution withdifferent volumes 1.5 and 2.0 ml. Plasma was directly generated from 5ml of stabilized or unstabilized blood samples. Residual blood wasstored for additional 6 days at room temperature before plasmageneration. ccfDNA was purified from 2 ml plasma, copy numbers of 18SrDNA gene were determined in triplicates by real time PCR.

Composition of stabilization reagent mixtures (for 10 ml K2EDTA wholeblood each):

-   -   0.5% PEG6000, 2.5 or 5% PEG300, DMPA, EDTA, Q-VD-OPh 2 ml: 60 mg        PEG6000, 300 or 600 μl PEG300, 120 μl DMPA, 162 mg K2EDTA, 12 μl        Q-VD-OPh (1 mg dissolved in 388 μl DMSO), water ad 2 ml    -   0.5% PEG6000, 2.5 or 5% PEG300, DMPA, EDTA, Q-VD-OPh 1.5 ml:        57.5 mg PEG6000, 287.5 μl or 575 μl PEG300, 115 μl DMPA, 155 mg        K2EDTA, 11.5 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), water        ad 1.5 ml

Thereby, the following final concentrations of the different componentsin the mixture are obtained after contact with blood:

-   -   unstabilized: 1.8 mg/ml K2EDTA    -   PEG6000, PEG300, DMPA, EDTA, Q-VD-OPh 2 ml: 0.5% (w/v) PEG6000,        2.5 or 5% (v/v) PEG300, 1% (v/v) DMPA, 15 mg/ml K2EDTA, 5 μM        Q-VD-OPh

Results

FIG. 12 depicts the results of quantitative real time PCR analyses fromeight different donors as change of copy numbers (average fold change)of the two tested 18S rDNA gene amplicons (66 bp or 500 bp) instabilized or unstabilized blood from the eight donors stored for 6 daysat room temperature to time point 0 (day 0) after blood draw. Allstabilized samples showed a comparable low increase of copy number foldchange of 18S rDNA gene amplicons. The achieved stabilization effect wasextraordinarily high as in all cases, the increase in the 500 bpfragment was far below 2-fold and was even below 1.5-fold even thoughthe samples were stored for a prolonged stabilization period of 6 days.

The respective stabilization compositions were also tested for change ofabsolute copy numbers of 18S rDNA amplicons at day 0 after blood draw inorder to analyse whether the detectable ccf DNA gene copy numbers isreduced. FIG. 13 shows the results. As can be seen, all testedstabilization compositions show an absolute copy number that iscomparable or even better compared to the unstabilized sample.Therefore, the nucleic acid yield was not reduced when using a silicacolumn based nucleic acid isolation approach for isolating the nucleicacids from the stabilized samples. The reduction in nucleic acid yieldobserved when a high molecular weight polyethylene glycol was used inhigher concentrations was not seen when using a combination of a highmolecular weight polyethylene glycol with a low molecular weightpolyethylene glycol. Using a respective combination allows to reduce theconcentration of high molecular weight polyethylene glycol withoutcompromising the stabilization effect which is supported by the lowmolecular weight polyethylene glycol. The low molecular weightpolyethylene glycol can also be used in higher concentrations withoutimpairing a subsequent nucleic acid isolation procedure that involves asilica column. The elevated standard deviations observed areattributable to the fact that there are variations in the amount ofccfDNA from donor to donor.

This demonstrates that a combination of a high molecular weightpolyethylene glycol in combination with a low molecular weightpolyethylene glycol is highly advantageous with respect to the achievedstabilization effect and the yield of nucleic acids that can be isolatedfrom the stabilized samples. Therefore, mixtures of differentpolyethylene glycols can be used in combination to efficiently stabilizethe blood samples without a reduction of absolute ccfDNA copy numbers.

13. Example 13

In example 13 the stabilization effect of a combination of a highmolecular weight PEG (0.5% PEG6000) and a low molecular weight PEG (2.5%or 5% PEG300) combined in aqueous stabilization solutions with BA, EDTAand a caspase inhibitor (Q-VD-OPh) were analyzed. Blood incubated with astabilization solution comprising a mixture of two amides (DMPA, BA),EDTA and a caspase inhibitor (Q-VD-OPh) was co-analyzed. EDTA bloodserved as unstabilized reference control.

Blood Collection and Stabilization

Samples of 10 ml whole blood from eight donors, collected in 10 ml spraydry K2EDTA tubes, were stabilized with mixtures of PEG300 and PEG6000,BA, EDTA and caspase inhibitor (Q-VD-OPh) in an aqueous solution with avolume of 1.5 ml. Plasma was directly generated from 5 ml of stabilizedor unstabilized blood samples. Residual blood was stored for additional6 days at room temperature before plasma generation. ccf DNA waspurified from 2 ml plasma, copy numbers of 18S rDNA gene were determinedin triplicates by real time PCR.

Composition of stabilization reagent mixtures (for 10 ml K2EDTA wholeblood each):

-   -   BA, DMPA, EDTA, Q-VD-OPh: 180 mg BA, 180 μl DMPA, 68.4 mg        K2EDTA, 12 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), water ad        2 ml    -   0.5% PEG6000, 2.5 or 5% PEG300, BA, EDTA, Q-VD-OPh 1.5 ml: 57.5        mg PEG6000, 287.5 μl or 575 μl PEG300, 115 mg BA, 155 mg K2EDTA,        11.5 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), water ad 1.5        ml

Thereby, the following final concentrations of the different componentsin whole blood/stabilization mixtures were obtained:

-   -   unstabilized: 1.8 mg/ml K2EDTA    -   BA, DMPA, EDTA, Q-VD-OPh: 1.5% (w/v) BA, 1.5% (v/v) DMPA, 7.2        mg/ml K2EDTA, 5 μM Q-VD-OPh    -   PEG6000, PEG300, BA, EDTA, Q-VD-OPh 1.5 ml: 0.5% (w/v) PEG6000,        2.5 or 5% (v/v) PEG300, 1% (w/v) BA, 15 mg/ml K2EDTA, 5 μM        Q-VD-OPh

Results

FIG. 14 shows the results of qPCR analyses from eight donors as averagechange of copy numbers (fold change) of the tested 66 bp or 500 bp long18S rDNA gene amplicons in stabilized or unstabilized blood from thedonors stored for 6 days at room temperature to time point 0 (day 0)after blood withdrawal. Whereas the unstabilized EDTA blood controlmanifested an elevation in average fold change in copy numbers, allstabilization compositions containing PEG showed only a low increasewith respect to the average fold change of 18S rDNA gene amplicons copynumbers. The results indicate a similar stabilization capability of thetested PEG containing stabilization compositions. The achievedstabilization was superior to the stabilization compositions comprisingBA, DMPA, EDTA and the caspase inhibitor thereby again demonstrating theimportant advantages that are achieved with the invention.

Additionally, it was confirmed that the described advantageousstabilization capability of the used stabilization solutions withcombinations of high and low molecular weight PEG is not accompanied bya reduction of ccfDNA copy numbers. This was analyzed by testing thesame solutions for change of absolute copy numbers of 18S rDNA ampliconsat day 0 in plasma after blood draw. FIG. 15 shows the obtained results.The obtained absolute copy number was similar to the stabilizationcomposition comprising BA, DMPA, EDTA and the caspase inhibitor.Therefore, no significant reduction in the absolute ccf DNA copy numberwas detected in this assay. Thus, combinations of different molecularweight PEGs, particularly of a high and low molecular weight PEG can becombined in different volumes of aqueous solutions containing BA toeffectively stabilize the extracellular nucleic acid population of bloodsamples, in particular by stabilizing white blood cells withoutsignificant reduction of absolute ccfDNA copy numbers when using astandard nucleic acid isolation procedure involving a silica membrane.

14. Example 14

In example 14, the effect of aqueous stabilization solutions was testedby hemolysis assays. Here, a combination of a high molecular weight PEG(0.5% PEG6000) and a low molecular weight PEG (2.5% or 5% PEG300) incombination with BA or DMPA and EDTA and caspase inhibitor (Q-VD-OPh)was analyzed. Blood incubated with a stabilization solution comprising amixture of two amides (DMPA, BA), EDTA and a caspase inhibitor(Q-VD-OPh) was co-analyzed. EDTA blood served as unstabilized referencecontrol.

Blood Collection and Stabilization

Samples of 10 ml whole blood from eight donors, collected in 10 ml spraydry K2EDTA tubes, were stabilized with mixtures of PEG300 and PEG6000,BA or DMPA, EDTA and caspase inhibitor (Q-VD-OPh) in an aqueous solutionwith a volumes of 1.5 ml. Plasma was directly generated from 5 ml ofstabilized or unstabilized blood samples. Residual blood was stored foradditional 6 days at room temperature before plasma generation.Hemoglobin content was determined by measuring absorbance at 414 nm on aspectrophotometer.

Composition of stabilization reagent mixtures (for 10 ml K2EDTA wholeblood each):

-   -   BA, DMPA, EDTA, Q-VD-OPh: 180 mg BA, 180 μl DMPA, 68.4 mg        K2EDTA, 12 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), water ad        2 ml    -   0.5% PEG6000, 2.5 or 5% PEG300, DMPA, EDTA, Q-VD-OPh: 57.5 mg        PEG6000, 287.5 μl or 575 μl PEG300, 115 μl DMPA, 155 mg K2EDTA,        11.5 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), water ad 1.5        ml    -   0.5% PEG6000, 2.5 or 5% PEG300, BA, EDTA, Q-VD-OPh: 57.5 mg        PEG6000, 287.5 μl or 575 μl PEG300, 115 mg BA, 155 mg K2EDTA,        11.5 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), water ad 1.5        ml

Thereby, the following final concentrations of the different componentsin whole blood/stabilization mixtures were obtained:

-   -   unstabilized: 1.8 mg/ml K2EDTA    -   BA, DMPA, EDTA, Q-VD-OPh: 1.5% (w/v) BA, 1.5% (v/v) DMPA, 7.2        mg/ml K2EDTA, 5 μM Q-VD-OPh    -   PEG6000, PEG300, DMPA, EDTA, Q-VD-OPh: 0.5% (w/v) PEG6000, 2.5        or 5% (v/v) PEG300, 1% (v/v) DMPA, 15 mg/ml K2EDTA, 5 μM        Q-VD-OPh    -   PEG6000, PEG300, BA, EDTA, Q-VD-OPh: 0.5% (w/v) PEG6000, 2.5 or        5% (v/v) PEG300, 1% (w/v) BA, 15 mg/ml K2EDTA, 5 μM Q-VD-OPh

Results

FIG. 16 shows the effect of the analyzed aqueous solutions comprisingdifferent molecular PEGs with either DMPA or BA on hemolysis in plasmasamples from 8 different donors after 0 and 6 days of blood storageafter draw. FIG. 16 shows the increase of hemolysis as increase ofabsorbance at 414 nm in the plasma fraction after 0 and 6 days of bloodstorage.

As can be seen in FIG. 16, EDTA control experiments reveal an increasein hemolysis (increase of absorption at 414 nm) after 6 days of bloodsample storage compared to the initial time point at 0 days afterstorage. With the stabilization compositions comprising mixtures of 0.5%PEG6000 (higher molecular PEG) and a PEG300 concentration of 5% (lowermolecular PEG) in combination with EDTA, caspase inhibitor (Q-VD-OPh)and BA or DMPA, hemolysis was similar to the EDTA reference sample. Incontrast, a stabilization composition according to the present inventionusing lower concentrations of of PEG300 (here: 2.5%) in combination with0.5% PEG6000, EDTA, caspase inhibitor (Q-VD-OPh) and BA or DMPA reducedhemolysis following blood storage for 6 days. This demonstrates thathemolysis may be prevented in stabilization compositions containing abalanced composition of high and low molecular weight PEG when dissolvedin an aqueous solution.

15. Example 15

A stabilization composition comprising PEG6000, BA, EDTA and a caspaseinhibitor (Q-VD-OPh) pre-filled in vacuumized blood collection tubes(Alpha tubes) was compared to commercially available Streck Cell-FreeDNA BCT tubes which comprise a stabilization composition that is basedon the use of a formaldehyde releaser as stabilizing agent.

Blood Collection and Stabilization

Samples of 10 ml whole blood from eight donors were collected in 10 mlspray dry K2EDTA, Alpha tubes pre-filled with amounts of stabilizationcomposition of the invention comprising PEG6000, EDTA, a caspaseinhibitor (Q-VD-OPh) and BA or DMPA and in Streck Cell-Free DNA BCTtubes. Plasma was directly generated from 5 ml of stabilized orunstabilized blood samples. Residual blood was stored for additional 3,6 and 10 days at room temperature before plasma generation. ccfDNA waspurified from 2 ml plasma, copy numbers of 18S rDNA gene were determinedin triplicates by real time PCR.

Composition of stabilization reagent mixtures in different tubes (allwith a draw volume of 10 ml blood):

-   -   EDTA 10 ml spray dried EDTA    -   Alpha1-Tube (PEG6000, BA, EDTA, Q-VD-OPh): 137.5 mg PEG6000, 55        mg BA, 165 mg K2EDTA, 11 μl Q-VD-OPh (1 mg dissolved in 388 μl        DMSO), water ad 1.0 ml    -   Alpha2-Tube (PEG6000, DMPA, EDTA, Q-VD-OPh): 110 mg PEG6000, 110        μl DMPA, 147 mg K2EDTA, 11 μl Q-VD-OPh (1 mg dissolved in 388 μl        DMSO), water ad 1.0 ml    -   Streck Cell-Free DNA BCT tube: comprises a formaldehyde releaser        as stabilizer

Thereby, the following final concentrations of the different componentsin whole blood/stabilization mixtures were obtained:

-   -   EDTA tube: 1.8 mg/ml K2EDTA    -   Alpha1-tube (PEG6000, BA, EDTA, Q-VD-OPh): 1.25% (w/v) PEG6000,        0.5% (w/v) BA, 15 mg/ml K2EDTA, 5 μM Q-VD-OPh    -   Alpha2-tube (PEG6000, DMPA, EDTA, Q-VD-OPh): 1.0% (w/v) PEG6000,        1.0% (v/v) DMPA, 15 mg/ml K2EDTA, 5 μM Q-VD-OPh    -   Streck Cell-Free DNA BCT tube: concentrations not applicable

Results

The results are shown in FIGS. 17 and 18. The change in copy numbers(average fold change) of 66 bp fragment (FIG. 17) and 500 bp fragment(FIG. 18) of the 18S rDNA gene in stabilized or unstabilized blood from8 donors stored for 3, 6 or 10 days at room temperature relative to timepoint 0 (day 0) after blood draw from the eight blood donors wasanalyzed. Bars indicate the corresponding standard deviation of theaverage fold change of the copy numbers from the eight donors percondition. As shown in FIGS. 17 and 18, both tested stabilizationcompositions comprising PEG6000, EDTA, a caspase inhibitor (Q-VD-OPh)and either BA or DMPA are highly efficient in stabilizing theextracellular nucleic acid population in blood. The average fold changeof copy numbers of the 66 bp fragment of the 18S rDNA stayed on thebasal level of time point zero for all tested time points (fold changeat around 1.0). FIG. 18 shows comparable results for the 500 bp fragmentlevels of the 18S rDNA gene i.e. the test fragment for cellular nucleicacids released during cell breakage substantially stayed at the statusof time point zero over the tested time period. This stabilizationeffect was comparable to the results where Streck Cell-Free DNA BCTtubes were used. Thus, the stabilization composition according to thepresent invention efficiently stabilizes the extracellular nucleic acidpopulation in a blood sample by reducing the release from intracellularnucleic acids such as in particular genomic DNA from white blood cellssimilar to Streck Cell-Free DNA BCT tubes which comprises formaldehydereleasers. However, as explained above, the use offormaldehyde-releasing substances has drawbacks, as they compromise theefficacy of extracellular nucleic acid isolation by induction ofcross-links between nucleic acid molecules or between proteins andnucleic acids. Therefore, specific nucleic acid isolation methods mustbe used. The stabilization composition according to the invention whichis based on the use of a poly(oxyethylene) polymer which does notinvolve the use of such cross-linking substances has importantadvantages over cross-linking based stabilization techniques.

16. Example 16

The stabilization effect of different high molecular weight PEGs (0.5%PEG6000, PEG10000 or PEG20000) in combination with a low molecularweight PEG (3.5% PEG300) combined in aqueous stabilization solutionswith BA or DMPA, EDTA and a caspase inhibitor (Q-VD-OPh) were analyzed.The stabilization additives were prefilled in vacuumized bloodcollection tubes (Alpha tubes). EDTA blood served as unstabilizedreference control.

Blood Collection and Stabilization

Samples of 10 ml whole blood from eight donors were collected in 10 mlspray dry K2EDTA tubes, and in Alpha tubes pre-filled with amounts ofstabilization composition of the invention comprising either PEG6000,PEG10000 or PEG20000 and additionally PEG300, EDTA, a caspase inhibitior(Q-VD-OPh) and BA or DMPA. Plasma was quickly generated from 5 ml ofstabilized or unstabilized blood samples. Residual blood was stored foradditional 6 days at room temperature before plasma generation. ccfDNAwas purified from 2 ml plasma, copy numbers of 18S rDNA gene weredetermined in triplicates by real time PCR.

Composition of stabilization reagent mixtures in different tubes (allwith a draw volume of 10 ml blood).

-   -   EDTA (unstabilized) 18 mg spray dried EDTA    -   Alpha3-Tube (0.5% PEG6000, 3.5% PEG300, DMPA, EDTA, Q-VD-OPh):        57.5 mg PEG6000, 402.50 PEG300, 115 μl DMPA, 152 mg K2EDTA, 11.5        μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), water ad 1.5 ml    -   Alpha4-Tube (0.5% PEG6000, 3.5% PEG300, BA, EDTA, Q-VD-OPh):        57.5 mg PEG6000, 402.5 μl PEG300, 115 μg BA, 152 mg K2EDTA, 11.5        μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), water ad 1.5 ml    -   Alpha5-Tube (0.5% PEG10000, 3.5% PEG300, DMPA, EDTA, Q-VD-OPh):        57.5 mg PEG10000, 402.5 μl PEG300, 115 μl DMPA, 152 mg K2EDTA,        11.5 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), water ad 1.5        ml    -   Alpha6-Tube (0.5% PEG10000, 3.5% PEG300, BA, EDTA, Q-VD-OPh):        57.5 mg PEG10000, 402.50 PEG300, 115 μg BA, 152 mg K2EDTA, 11.5        μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), water ad 1.5 ml    -   Alpha7-Tube (0.5% PEG20000, 3.5% PEG300, DMPA, EDTA, Q-VD-OPh):        57.5 mg PEG20000, 402.5 μl PEG300, 115 μl DMPA, 152 mg K2EDTA,        11.5 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), water ad 1.5        ml    -   Alpha8-Tube (0.5% PEG20000, 3.5% PEG300, BA, EDTA, Q-VD-OPh):        57.5 mg PEG20000, 402.50 PEG300, 115 μg BA, 152 mg K2EDTA, 11.5        μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), water ad 1.5 ml

Thereby, the following final concentrations of the different componentsin whole blood/stabilization mixtures were obtained:

-   -   unstabilized: 1.8 mg/ml K2EDTA    -   PEG6000, PEG10000 or PEG20000, PEG300, DMPA or BA, EDTA,        Q-VD-OPh 1.5 ml: 0.5% (w/v) PEG6000 (or PEG10000, or PEG20000),        5.5% (v/v) PEG300, 1% (v/v) DMPA or (w/v) BA, 13.2 mg/ml K2EDTA,        5 μM Q-VD-OPh

Results

The results are shown in FIGS. 19 and 20. FIG. 19. demonstrates thechange in copy numbers (average fold change) of 66 bp or 500 pb fragmentof the 18S rDNA gene in stabilized or unstabilized blood from 8 donorsstored for 6 days at room temperature relative to time point 0 (day 0).FIG. 20 shows the absolute copy numbers at day 0 in plasma, directlyafter the blood draw. As shown in FIG. 19, PEGs with a molecular weightof up to 20.000 in compositions comprising additionally PEG300, EDTA, acaspase inhibitor (Q-VD-OPh) and either BA or DMPA are highly efficientin stabilizing the extracellular nucleic acid population in blood. Incontrast to unstabilized EDTA blood, for the stabilized blood from AlphaTubes 3-8 the average fold change of copy numbers for both the 66 and500 bp fragment of the 18S rDNA stayed on the basal level of time pointzero after 6 days storage (fold change at around 1.0). In addition, thebalanced compositions of high and low molecular weight PEGs did notreduce the absolute copy numbers of ccfDNA in comparison to plasma fromunstabilized EDTA directly after blood draw (see FIG. 20), therebydemonstrating that extracellular nucleic acids could be effectivelyisolated from the stabilized samples. That the absolute copy numbers arelower in the stabilized samples compared to the unstabilized referencesample is attributable to the fact that the obtained plasma is dilutedwith the liquid stabilization composition. Due to this dilution, theinitial amount of nucleic acids is lower in the stabilized samples.

17. Example 17

The stabilization technology of the present invention is also compatiblewith an anion exchange based nucleic acid isolation protocol.Extracellular nucleic acids were isolated from plasma that was obtainedfrom a stabilized blood sample. Stabilization was performed using astabilization composition comprising K₂EDTA, Q-VD-OPH, DMPA, PEG 10000,PEG300 in water (see above). Extracellular nucleic acids were isolatedfrom the stabilized plasma samples using the QIAamp circulating nucleicacid kit (QIAGEN) according to the manufacturers instructions (2 mlplasma volume, 60 μl elution volume). Alternatively, extracelluarnucleic acids were isolated from stabilized plasma samples (2 ml) usingmagnetic particles comprising anion exchange groups (tertiary aminegroups) as solid phase for nucleic acid binding. The sample wasdisrupted and binding occurred at an acidic pH (4.5). The bound nucleicacids were washed three times and eluted using 75 μl of an alkalineelution buffer (pH 12.5). The protocol was performed using an automatedsystem (QIAsymphony).

The nucleic acid yield (18s rDNA (66 bp) and 18s rDNA (500 bp) obtainedwith the anion exchange based nucleic acid protocol was compared to theresults obtained with the QIAamp circulating nucleic acid kit (set as100%) by PCR analysis (compared to a genomic DNA dilution series todetermine the copy number). The subsequent table shows the results:

Anion exchange based nucleic QIAamp circulating acid isolation protocolnucleic acid kit 18srDNA (66bp) 98.64 100.00 18s rDNA 88.04 100.00(500bp)

As can be seen, extracellular nucleic acids could be obtained with highyield from the stabilized samples using an anion exchange based nucleicacid isolation protocol.

1. A method for stabilizing an extracellular nucleic acid populationcomprised in a cell-containing biological sample comprising contactingthe cell-containing biological sample with at least onepoly(oxyethylene) polymer as stabilizing agent or with mono-ethyleneglycol as stabilizing agent.
 2. The method according to claim 1, whereinthe poly(oxyethylene) polymer is polyethylene glycol.
 3. The methodaccording to claim 1, wherein the poly(oxyethylene) polymer is a highmolecular weight poly(oxyethylene) polymer having a molecular weight ofat least
 1500. 4. The method according to claim 3, wherein the highmolecular weight poly(oxyethylene) polymer has a molecular weight thatlies in a range selected from 1500 to 50000, 2000 to 40000, 2500 to30000, 2500 to 25000, 3000 to 20000 and 3500 to
 15000. 5. The methodaccording to claim 3, wherein after the cell-containing biologicalsample has been contacted with the high molecular weightpoly(oxyethylene) polymer and optionally further additives used forstabilization, the resulting mixture comprises the high molecular weightpoly(oxyethylene) polymer in a concentration range that is selected from0.05% to 4% (w/v), 0.1% to 3% (w/v), 0.2% to 2.5% (w/v), 0.25% to 2%(w/v), 0.3% to 1.75% (w/v) and 0.35% to 1.5% (w/v) or is selected from0.25% to 1.5% (w/v), 0.3% to 1.25% (w/v), 0.35% to 1% (w/v) and 0.4% to0.75% (w/v).
 6. The method according to claim 1, wherein thepoly(oxyethylene) polymer has a molecular weight below 1500 andpreferably is a low molecular weight poly(oxyethylene) polymer having amolecular weight of 1000 or less.
 7. The method according to claim 6,wherein the poly(oxyethylene) polymer is a low molecular weightpoly(oxyethylene) polymer having a molecular weight of 1000 or less andpreferably, the molecular weight lies in a range selected from 100 to800, 150 to 700, 200 to 600 and 200 to
 500. 8. The method according toclaim 6, wherein after the cell-containing biological sample has beencontacted with the poly(oxyethylene) polymer and optionally furtheradditives used for stabilization, the resulting mixture comprises thepoly(oxyethylene) polymer, such as a low molecular weightpoly(oxyethylene) polymer having a molecular weight of 1000 or less, ina concentration range that is selected from 0.5% to 10%, 1.5% to 9%, 2%to 8%, 2 to 7%, 2.5% to 7% and 3% to 6%.
 9. The method according toclaim 3, wherein the cell-containing biological sample is contacted witha high molecular weight poly(oxyethylene) polymer having a molecularweight of at least 1500 and a low molecular weight poly(oxyethylene)having a molecular weight of 1000 or less.
 10. The method according toclaim 9, wherein after the cell-containing biological sample has beencontacted with the high molecular weight poly(oxyethylene) polymer andoptionally further additives used for stabilization, the resultingmixture comprises the high molecular weight poly(oxyethylene) polymer ina concentration range selected from 0.1% to 3% (w/v), 0.2% to 2.5%(w/v), 0.25% to 2% (w/v), 0.3% to 1.75% (w/v) and 0.35% to 1.5% (w/v) orselected from 0.25% to 1.5% (w/v), 0.3% to 1.25% (w/v), 0.35% to 1%(w/v) and 0.4% to 0.75% (w/v); and the low molecular weightpoly(oxyethylene) polymer in a concentration range selected from 0.5% to10%, 1.5% to 9%, 1.75% to 8%, 2% to 7% and 2.5% to 6%.
 11. The methodaccording to claim 1, wherein the cell-containing biological sample isblood and wherein the blood sample is additionally contacted with ananticoagulant, preferably a chelating agent.
 12. The method according toclaim 11, wherein the blood sample is contacted with a high molecularweight poly(oxyethylene) polymer having a molecular weight that lies ina range selected from 3000 to 40000, 2500 to 25000 and 4000 to 20000, alow molecular weight poly(oxyethylene) polymer having a molecular weightthat lies in a range selected from 200 to 800, 200 to 600 and 200 to 500and an anticoagulant, and wherein after the blood sample has beencontacted with the high and low molecular weight poly(oxyethylene)polymer, the anticoagulant and optionally further additives used forstabilization, the resulting mixture comprises the high molecular weightpoly(oxyethylene) polymer in a concentration that lies in a rangeselected from 0.2% to 1.5% (w/v), 0.3% to 1.25% (w/v) and 0.4 (w/v) to0.75% (w/v) and the low molecular weight poly(oxyethylene) polymer in aconcentration that lies in the range of 2% to 7%, preferably 2.25% to6%.
 13. The method according to claim 1, wherein for stabilization, thecell-containing sample is additionally contacted with one or moreprimary, secondary or tertiary amides and/or at least one caspaseinhibitor as stabilizing agent(s).
 14. The method according to claim 13,wherein the primary, secondary or tertiary amide is a compound accordingto formula 1

wherein R1 is a hydrogen residue or an alkyl residue, preferably a C1-C5alkyl residue, a C1-C4 alkyl residue or a C1-C3 alkyl residue, morepreferred a C1-C2 alkyl residue, R2 and R3 are identical or differentand are selected from a hydrogen residue and a hydrocarbon residue,preferably an alkyl residue, with a length of the carbon chain of 1-20atoms arranged in a linear or branched manner, and R4 is an oxygen,sulphur or selenium residue, preferably R4 is oxygen.
 15. The methodaccording to claim 1, wherein the cell-containing sample is contactedwith butanamide and/or an N,N-dialkylpropanamide, wherein saidN,N-dialkylpropanamide preferably is N,N-dimethylpropanamide.
 16. Themethod according to claim 1, wherein the cell-containing biologicalsample, which preferably is a blood sample or a sample derived fromblood such as plasma or serum, is contacted with: a) at least one highmolecular weight poly(oxyethylene) polymer having a molecular weight ofat least 1500, preferably in a range of 2000 to 40000, more preferred2000 to 30000, 2500 to 25000 or 3000 to 20000; b) one or more compoundsaccording to formula 1, preferably in a concentration so that theconcentration in the mixture with the cell-containing biological samplelies in a range of 0.25% to 5%, 0.3% to 4%, 0.4% to 3%, 0.5% to 2% or0.75% to 1.5%; c) at least one caspase inhibitor, preferably apancaspase inhibitor, more preferred Q-VD-OPh, preferably in aconcentration so that the concentration of the caspase inhibitor in themixture with the cell-containing biological sample lies in a range of0.1 μM to 20 μM, more preferred 0.5 μM to 10 μM, more preferred 1 μM to10 μM, more preferred 3 μM to 7.5 μM; d) optionally at least one furtherpoly(oxyethylene) polymer having a molecular weight that is at least100, preferably at least 200, at least 300 or at least 400 below themolecular weight of the high molecular weight poly(oxyethylene) polymerused and wherein said further poly(oxyethylene) polymer preferably is alow molecular weight poly(oxyethylene) having a molecular weight of 1000or less, preferably having a molecular weight in a range of 200 to 800or 200 to 600; and/or e) optionally a chelating agent, more preferablyEDTA.
 17. The method according to claim 14, wherein for stabilization,the cell-containing sample which preferably is a blood sample, iscontacted with: a) at least one high molecular weight poly(oxyethylene)polymer having a molecular weight of at least 3000; b) one or morecompounds according to formula 1; c) at least one caspase inhibitor; d)optionally at least one low molecular weight poly(oxyethylene) polymerhaving a molecular weight of 1000 or less; and/or e) optionally achelating agent, preferably EDTA, wherein the release of genomic DNAfrom cells contained in the cell-containing sample into the cell-freeportion of the sample is reduced due to the stabilization.
 18. Themethod according to claim 1, wherein the cell-containing sample is ablood sample which is contacted with: a) at least one high molecularweight poly(oxyethylene) polymer having a molecular weight that lies ina range of 3000 to 40000, 3000 to 30000 or 3500 to 25000; b) one or morecompounds according to formula 1; c) at least one caspase inhibitor,preferably a pancaspase inhibitor, more preferred Q-VD-OPh; d) at leastone low molecular weight poly(oxyethylene) polymer having a molecularweight of 1000 or less, preferably in a range of 100 to 800, 200 to 600or 200 to 500; and/or e) an anticoagulant which preferably is achelating agent, preferably EDTA, wherein after the blood sample hasbeen contacted with said additives and optionally further additives usedfor stabilization the resulting mixture comprises the high molecularweight poly(oxyethylene) polymer in a concentration that lies in a rangeof 0.2% to 1.5% (w/v), 0.25% to 1.25% (w/v), 0.3% to 1% (w/v) or 0.4% to0.75% (w/v), the one or more compounds according to formula 1 in aconcentration that lies in a range of 0.3% to 4%, preferably 0.5 to 3%,0.5 to 2% or 0.75 to 1.5%, the caspase inhibitor in a concentration thatlies in a range of 1 μM to 10 μM, preferably 3 μM to 7.5 μM, and the lowmolecular weight poly(oxyethylene) polymer in a concentration that liesin the range of 1.5% to 10%, preferably 2% to 6%.
 19. The methodaccording to claim 1, comprising contacting the cell-containingbiological sample with mono-ethylenglycol as stabilizing agent andwherein optionally, the cell-containing sample is additionally contactedwith at least one poly(oxyethylene) polymer; and/or wherein thecell-containing sample is additionally contacted with one or moreprimary, secondary or tertiary amides and/or at least one caspaseinhibitor.
 20. The method according to claim 1, wherein the compoundsused for stabilization are contained in an stabilization compositioncomprising water.
 21. The method according to claim 1, having one ormore of the following characteristics: i) the stabilization does notinvolve the use of additives in a concentration wherein said additiveswould induce or promote lysis of nucleated cells; ii) the stabilizationdoes not involve the use of a cross-linking agent that inducesprotein-nucleic acid and/or protein-protein crosslinks such asformaldehyde, formaline, paraformaldehyde or a formaldehyde releaser;and/or iii) the stabilization does not involve the use of toxic agents.22. The method of claim 1, further comprising isolating extracellularnucleic acids from the stabilized biological sample.
 23. A compositionsuitable for stabilizing a cell-containing biological sample, comprisingi) a poly(oxyethylene) polymer as stabilizing agent or ii) mono-ethyleneglycol as stabilizing agent and one or more further additives selectedfrom the group consisting of one or more primary, secondary or tertiaryamides; a caspase inhibitor; and an anticoagulant and/or a chelatingagent. 24.-33. (canceled)